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CHAPTER 1
INTRODUCTION
Antarctica – the most challenging living environment
The mechanism of life is often separated into three classes: growth, maintenance,
and survival (Morita 1997). However, the limitations of life are not well-defined or
understood and have primarily been advanced though exploration and discover of
organisms living in “extreme” environments where life was not thought possible.
Antarctica is a good example of “extreme” environment where microorganisms live in
subzero temperatures with the constraints include the reduction of available liquid water
accompanied by the formation of ice crystals, lower rates of catalysis and transport,
decreased membrane fluidity, and stability of molecular structures (Cavicchioli et al.,
2000). Recent exploration and investigations of low-temperature environments are
redefining the known limits of microbial activity and are fuelled by new techniques and
capabilities.
Due to the extremely harsh climatic conditions, Antarctica’s microbial ecology
and the techniques of survival for the microbial life are not well-studies. These studies
are important as Antarctica is a considerable reservoir of microbial diversity (Priscu &
Christner 2004). Antarctica is a cryosphere that not only act as an integral part of global
climate system but also as one of the major habitable ecosystems of Earth’s biosphere
which is the best analogue for the search of novel extraterrestrial life .
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Objectives of this research project
This project aims to isolate culturable bacteria from Antarctic soil samples. The
bacteria isolated from Antarctic soil samples will then be screened for quorum sensing
and quorum quenching abilities. The selected Antarctic isolates will be used to further
study their quorum sensing and quorum quenching systems.
2
CHAPTER 2
LITERATURE REVIEW
2.1 Bacteria cell-to-cell communication- Quorum Sensing (QS)
The bacteria world had been described by Francois Jacob (1973) as “The
universe without sex, without hormone and without nervous systems populated only by
individual cells reproducing ad infinitum”. Until 1990s, researchers found that bacteria
are actually highly communicative with each other and with their hosts or competitors
in the same living environment. Fuqua et al., 1994 used the term ‘quorum sensing’ (QS)
to describe the bacteria highly social world via chemical communication, and the
capability of sophisticated co-operative behaviour (Williams et al., 2007a). Bacteria in
fact are excellent linguist; they are able to have secret talk among their own kind, by
producing species-specific chemical signalling molecules. For example, N-acylhomoserine lactone (AHL) in gram negative bacteria and small peptides in gram
positive bacteria (William et al., 2007b).
They produce autoinducer-2 (AI-2), which allows them to talk to different
species. This mechanism needs an evolution of a sophisticated QS circuit, to detect and
integrate the information contains in multiple autoinducers (Lerat & Moran 2004). A
few studies also indicated bacteria even have cross-kingdom communication with their
eukaryotes host (Williams et al., 2007a).
QS is a gene regulatory mechanism, which is cell population density-dependent
and is mediated by self generated extracellular signal molecules (Williams & Camera
2009). Different families of signal molecules have been identified in gram positive and
gram negative bacteria. QS regulates bioluminescence, virulence factor expression,
biofilm formation, sporulation, mating, production of antibiotic and other phenotypes
3
(Miller & Bassler, 2001). All these behaviours are unproductive when undertaken by an
individual bacterium but become effective by the simultaneous action of a group of cells.
This cell density dependant regulatory system relies on the accumulation of
autoinducers that secreted by bacterium to extracellular environment, and when they
reach a threshold level, the autoinducers will diffuse back into the bacteria, bind to the
regulatory protein and regulate the gene expression (Williams et al., 2007).
2.1.1 QS in Gram negative bacteria
AHLs are conserved QS signal molecules produced by a range of gram negative
bacterial species. It contains a conserved homoserine lactone (HSL) ring unsubstituted
in the β- and γ-positions which is N-acylated with a fatty acyl group at the α-position
(Chhabra et al., 2005) and an amide (N)-linked acyl side chain which differ in length
(range from 4-18 carbons), degree of saturation, and the presence of C-3 substituent
(could be hydroxyl- or oxo-) (Fuqua et al., 2001, Swift et al., 2003). These AHLs are
synthesized by gram negative bacteria via enzymes belonging to the LuxI or LuxM
protein familes and control the gene expression by interact with LuxR family of
response regulator protein (Zhu & Winans, 2001).
AHLs are involved in the induction of virulent genes in pathogens such as
Pseudomonas aeruginosa (Passador et al., 1993), bioluminescence in Vibrio species
(Cao et al., 1989, Dunphy et al., 1997), and development of biofilm in microbial
biofilm populations (Davies et al, 1998). More bacterial species have been discovered
to produce AHLs, but their biological functions remain unclear. AHLs produced by
LuxI synthase, bind to its cognate receptor (LuxR) which in turn will regulate genes
expression and each LuxR-type protein is highly selective for its cognate AHL signal
molecule.
4
There are more than 50 species gram negative bacteria produced AHL and these
bacteria belong to genera inhabiting a wide variety of environmental niches – from
marine and freshwater environments to soil, plants and animals, including many
pathogens, symbionts, plant-growth promoting bacteria and extremophiles . Examples
include Acidithiobacillus, Acinetobacter, Aeromonas, Agrobacterium, Brucella,
Burkholderia, Erwinia, Enterobacter, Chromobacterium, Hafnia, Mesorhizobium,
Methylobacter, Paracoccus, and Pseudomonas. Farrah et al., 2005, did described in
extreme acidic environment (pH 2), Acidithiobacillus ferrooxidans produce medium
and long chain AHLs with different C3-substitution and could be part of a regulon
controlling some physiological functions for example biofilm formation. This would
suggest that other extreme environment such as hot spring, soda lake or soda deserts,
deep sea may have other extremophiles to produce AHLs to regulate genes expressions.
Figure 2.1 Chemical structures of some AHLs
(http://www.pharmainfo.net/reviews/quorum-sensing-communication-between-bacteria)
Abbreviations : C4-HSL, N-(butanoyl)-L-homoserine lactone; C6-HSL, N-(hexanoyl)-Lhomoserine lactone; C8-HSL, N-(octanoyl)-L-homoserine lactone; C10-HSL, N(decanoyl)-L-homoserine lactone; C12-HSL, N-(docecanoyl)-L-homoserine lactone;
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C14-HSL, N-(tetradecanoyl)-L-homoserine lactone; 3-hyroxy-C4-HSL, N-(3hydroxybutanoyl)-L-homoserine lactone; 3-oxo-C6-HSL, N-(3-oxohexanoyl)-Lhomoserine lactone; 3-oxo-C8-HSL, N-(3-oxooctanoyl)-L-homoserine lactone; 3-oxoC10-HSL, N-(3-oxodecanoyl)-L-homoserine lactone; 3-oxo-C12-HSL, N-(3oxodocecanoyl)-L-homoserine lactone; 3-oxo-C14-HSL, N-(3-oxotetradecanoyl)-Lhomoserine lactone.
Table 2.1 Examples of organisms with homologues of the luxI and luxR genes
Bacteria
LuxR/LuxI
AHLs
Phenotype
Reference
C4-HSL
Extracellular
Swift et al., 1997
homolougues
Aeromonas
AsaR/I
salmonicida
Agrobacterium
protease
TraR/I
tumefaciens
3-oxo-C8-
Conjugation
Piper et al., 1999
HSL
Burkholderia
CepR/I,
C6-HSL,
Biofilm
Tomlin et al.,
cenocepacia
CciR/I
C8-HSL
formation
2004
3-oxo-C6-
Carbapenem
Bainton et al.,
HSL
antibiotic,
1992; Pirhonen
exoenzymes
et al., 1993
3-oxo-C6-
Adhesion and
Koutsoudis et al.,
HSL
host
2006
Erwinia carotovora CarR, ExpR
Subsp carotovora
ExpI(CarI)
Pantoea stewartii
EsaR/I
colonization
Pseudomonas
LasR/I
3-oxo-
Exoenzymes,
Chapon-Herve et
6
aeruginosa
C12-HSL
biofilm
al., 1997,
formation, cell-
Gambello &
cell spacing
Iglewski, 1991,
Passador et al.,
1993, Glessner et
al., 1999
Pseudomonas
PhzR/I
fluorescens
Pseudomonas
PpuR/I
3-
Phenazine
Shaw et al.,
hydroxy-
antibiotic
1997; Khan et al.,
C6-HSL
production
2007
3-oxo-
Maturation
Dubern et al.,
C10-HSL,
putida
2006
3-oxoC12-HSL
Rhizobium
RhiR/I
leguminosarum
Rhodobacter
CerR/I
sphaeroides
Serratia
SwrR/I
3-
Nodulation,
Gray, 1997;
hydroxy-
bacteriocin,
Rodelas et al.,
7-cis-C14-
stationary
1999; Thorn &
HSL
phase survival
Williams, 1999
7-cis-C14-
Community
Puskas et al.,
HSL
escape
1997
C4-HSL
Biofilm
Labbate et al.,
formation
2004
3-oxo-C6-
Motility,
Atkinson et al.,
HSL
clumping
1999
liquefaciens
Yersinia
pseudotuberculosis
YpsR/I
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2.1.2 QS in bacteria in extreme environment
Acidithiobacillus ferrooxidans was the first extremophile that was reported to
produce signalling molecules (AHLs). This bacterium that lives in
extreme
environment of pH2, produce a range of long chain AHLs such as C12-HSL, C14-HSL,
3-oxo-C14-HSL, 3-hydroxy-C8-HSL, , 3-hydroxy-C10-HSL, 3-hydroxy-C12-HSL, 3hydroxy-C14-HSL, and 3-hydroxy-C16-HSL (Farah et al., 2005, Rivas et al., 2005,
Rivas et al., 2007, Ruis et al., 2008). However, the target gene and function of the
signalling molecules remain unknown.
Some facultative anaerobic bacteria produce AHLs as well, for example
Aeromonas spp. isolated from Malaysian patient (Chan et al., 2010). The
characterisation of the AHLs produced by Aeromonas spp. using thin layer
chromatography overlaid with biosensor CV026 showed the production of C4- and 3Oxo-C6-HSL. However, the studies on the production and characterization of the AHLs
produced by these Aeromonas spp. were done in aerobic condition and the production
of this QS signal in anaerobic condition remains unknown.
2.1.3 Production of 4-Quinolone Signal in Pseudomonas spp.
QS system of Pseudomonas species such as Pseudomonas aeruginosa are more
complicated as compared with other bacteria. In these bacteria, a class of 4-Quinolone
signal was produced to regulate numerous virulence genes and other QS-regulated traits.
In Pseudomonas aeruginosa, the production of 2-heptyl-3-hydroxy-4(1H)-quinolones,
the Pseudomonas quinolone signal (PQS) regulates certain virulence genes including
iron scavenging (Diggle et al., 2007). PQS also regulates the production of other AHLs
production including 3-oxo-C12-HSL. In other Pseudomonas species, precursor of PQS,
2-heptyl-4(1H)-quinolone (HHQ) was produced, and Diggle et al., 2003 show that
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HHQ act as an QS signal per se. The production of pyocyanin is affected in the mutant
of these QS system and production of pyocyanin cannot be restored with
supplementation of PQS.
However, pseudomonad is not the only bacterium that produces HHQ.
Burkholderia pseudomallei, also produces HHQ as a QS signal, but Burkholderia
pseudomallei lack the gene that converts the HHQ to PQS (pqsH homolog). Recently a
few Burkholderia species, for example, B. thailandensis, and B. ambifaria produce 4Hydroxy-2-Alkylquinoline Analogues with a methyl group at the 3-position that is
required for QS regulation (Vial et al., 2008).
2.2 Quorum Quenching- breakdown of the communication
Signalling molecules can be quenched to block the communication between the
members of bacterial population. This mechanism is called quorum quenching (QQ).
Plenty of bacteria and eukaryotic organisms have been reported to be able to inactivate
AHLs molecules, Gram positive bacteria such as Rhodococcus erythropolis is capable
of using AHLs as the sole carbon and energy source (Uroz et al., 2005), AHL
inactivation activity was found serum samples of mammalians (Yang et al., 2005).
Production of AHL antagonists has also been demonstrated in marine red algae
(Givskov et al., 1996).
AHLs can be inactivated enzymically with two families of enzymes have been
reported so far, namely AHL lactone hydrolases and AHL acylases (Dong et al., 2000,
Lin et al., 2003). QQ has great potential in combating pathogenic bacteria. QQ
mechanisms play important roles in microbe–microbe and pathogen–host interactions
and have been used, or served as lead compounds, in developing and formulating a new
generation of antimicrobials (Dong et al., 2007). Reports over the past few years also
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demonstrated that QQ mechanisms are widely conserved in many prokaryotic and
eukaryotic organisms (Table 2.2).
In 2000, the first QQ enzymes was reported by Dong et al., the bacteria that
produce the enzyme was soil Bacillus species. The gene that produces QQ enzymes was
aiiA and the product was characterized as an AHL-lactonase (Dong et al., 2001). This
gram positive bacteria produce AiiA to hydrolase the ester bond of the lactone ring to
give acylhomoserine. The gene that produces this enzyme is highly conserved (Dong et
al., 2002).
In the same year, Leadbetter and Greenberg, reported another soil bacterium,
Variovorax paradoxus that utilized AHLs as sole carbon and nitrogen source. This
organism actually degrades the acyl-side chain that releases the homoserine lactone ring
and a carboxylic acid. The bacteria then utilize carboxylic acid in the absence of other
carbon source.
Table 2.2 Examples of QQ enzymes in prokaryotes and eukaryotes
Species
Gene
Enzyme
Reference
Bacillus sp. 240B1
aiiA
AHL lactonase
Dong et al., 2000
B. thuringiensis
aiiA
AHL lactonase
Dong et al., 2002;
AHL lactonase
Dong et al., 2002;
Prokaryotes
homologues
B.cereus
aiiA
homologues
Reimmann et al.,
2002
10
B.mycoides
aiiA
AHL lactonase
Dong et al.,2002
AHL lactonase
Ulrich, 2004
AHL lactonase
Zhang et al., 2002;
homologues
B.anthracis
aiiA
homologues
Agrobacterium
attM,aiiB
tumefaciens
Arthrobacter sp.
Carlier et al., 2003
ahlD
AHL lactonase
Park et al., 2003
ahlK
AHL lactonase
Park et al., 2003
ND
AHL acylase
Leadbetter &
IBN110
Klebsiella
pneumoniae
Variovorax
paradoxus VAI-C
Ralstonia strain
Greenberg, 2000
aiiD
AHL acylase
XJ12B
Pseudomonas strain
Lin et al., 2003; Hu
et al., 2003
pvdQ
AHL acylase
Huang et al., 2003
Not known
AHL aminohydrolase and Uroz et al., 2005
PAI-A (PAO1)
Rhodococcus
erythropolis W2
one unknown enzyme
Eukaryotes
Human (airway
epithelia)
PONs
Lactonase
Chun et al., 2003;
Greenberg et al.,
11
2004
Porcine (kidney)
ACY1
Acylase1
Xu et al., 2003
ND= not known
2.2.1 Applications of QQ in various fields
As shown in Table 1.1, there are diverse activities regulated by the QS system of
many bacteria. Thus the study of QQ is of pivotal importance especially when the QQ
targets the AHLs involved in the regulation of virulence factor that attack plants and
animals including human. For example, the expression of lactonase in plant showed
increased in resistance against plant pathogen, Erwinia carotovora (Dong et al., 2001).
Thus QQ has a potential to reduce plant pathogenesis due to bacterial infection in the
large scale production of plant especially crops. This seems to be a novel strategy to
produce crops of good quality by using this biocontrol agent and to meet the need of
increasing world population. At the same time, biosafety issue should also be
considered so as to avoid problems such as cross-contamination of other crops,
development of transgenic plant with unwanted characteristics and ecological problems.
The conventional therapy against bacterial infection generally depends on antimicrobial compounds aiming to kill the invaded pathogenic microbes. However as the
anti-microbial compounds have been used frequently, there is problem of some bacteria
being resistant to the antibiotics. Thus QQ which aims to interrupt the activities
regulated by QS especially virulence factor production seems to be a promising strategy
to attenuate the pathogens and to stop the actions. Eventhough recent research showed
that bacteria may evolve resistance to QS inhibitor brominated furanone C-30 (Maeda et
al., 2012), QQ enzyme still have great potential as alternative for antibiotics.
12
It is of a great value to study pathogens which cause many diseases to a range of
hosts, for example Pseudomonas sp. QS has been involved in the virulence of a few
Pseudomonas to cause a wide range of infections in humans. Pseudomonas aeruginosa
and Pseudomonas putida are opportunistic agents that can cause various infections in
immunocompromised patients. For example, Pseudomonas aeruginosa cause infections
like malignant external otitis, meningitis, pneumoniacystic fibrosis and so on whereas
Pseudomonas putida causes infections in patients with solid tumour (Bodey et al., 1983;
Martino, et al., 1996). Thus QQ which targets the QS in these bacteria is very important
for clinical purposes and there are many studies particularly on infections caused by
pseudomonads. For example azithromycin caused reduction in virulence factor
production by disrupting the QS system in Pseudomonas aeruginosa (Tateda et al.,
2001).
2.3 Importance of polar research-QS and QQ of Antarctic Bacteria
In Antarctica, the most challenging habitat on Earth, the coldest, driest, and
windiest continent, working in a community dependent manner will be more effective
than only a single bacteria cell. This phenomenon, where a bacterial population behaves
co-operatively and recognizes self from non-self, could be highly advantageous
particularly in the contexts of symbiosis and niche adaptation, production of secondary
metabolites, and for facilitating population migration if prevailing condition in a
specific environment niche have become unfavourable (Williams et al., 2008).
There have yet been any literatures describing QS and QQ activities in Antarctic
bacteria. However, from the biodiversity studies of certain locations in Antarctica,
including Signy Island (Chong et al., 2009b), as one of the sampling site of this studies,
showed a high proportion of γ-proteobacteria such as Acinetobacter spp. and
13
Pseudomonas spp. The mesophilic strains of all these bacteria have been reported to
utilise AHLs as their QS signal (Kang et al., 2004, Huang et al., 2003)
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CHAPTER 3
MATERIALS AND METHODS
3.1 Equipments
Autoclave machine (Hirayama), Rapid Resolution Liquid Chromatography
(Agilent, model 1200), Triple Quadrupole LC/MS (Agilent, model 6400), LC/MS ITTOF (Shimadzu), Luminometer (Tecan ,infinite 200) Thermal Cycles PCR machine
(Bio-rad, model 2700) Mini centrifuge (Eppendorf, mini spin), Thermomixer compact
(Eppendorf), Spectrophotometer (Shimadzu, model UV1601), Research inverted
microscope (Olympus, model 1X71-22FL/PH), Weighting machine (Sartorius), pH
meter (Cybersccan pH500), High Performance UV transilluminator (UVP), Water
distiller (Exelo), Ultra-pure water (Milipore), Refrigerated Orbital shaker incubator (NBIOTEK. INC.), Eyela oil bath micropipette and pipet tips.
3.2 Commercial kits
The commercial kits used for Gram staining, genomic DNA isolation, DNA
purification, and plasmid DNA isolation are as below:
(i)
BDTM Gram Stain Kit (Becton, Dickinson and Company, USA)
(ii)
QIAamp® DNA Mini Kit (Qiagen Pty. Ltd., Germany)
(iii)
QIAquick® Gel Extraction Kit (Qiagen Pty. Ltd., Germany)
(iv)
QIAquick® PCR Purification Kit (Qiagen Pty. Ltd., Germany)
(v)
QIAquick® Spin Miniprep Kit (Qiagen Pty. Ltd., Germany)
15
3.3 Soil Sampling
Sterile vessels or tube for samples collection were all pre-cooled. The soil
samples were collected from a few ecologically different sites. A few soil samples from
sampling sites located at Casey station area, Windwill Island were collected by Dr.
Chong Chun Wie. The samples were either processed immediately or kept in -20°C.
The other sampling sites of this studied were located at Rothera Research Station. The
locations and description of the sampling site are shown in Fig. 3.1, Table 3.1 (Casey
Research Station) Fig. 3.2, and Table 3.2 (Rothera Research Station).
Fig.3.1 Sampling locations in Casey Station area, Windwill Island which the soil
samples were collected by Dr. Chong Chun Wie. × indicates the sampling sites.
Indicates location of Casey research station and one of the sampling site Thala Valley.
Source:
Australian
Antarctic
Division
Data
Centre
(http://aadc-
maps.aad.gov.au/aadc/mapcat/index_new.cfm) and Chong et al., 2009a.
16
Table 3.1 The sampling sites of Casey Research Station for soil samples collected
by Dr. Chong Chun Wie (Chong et al., 2009a).
1
2
3
Site
Description
GPS
Thala Valley
Former waste disposal site. Approximately,
66º16’49’’S;
500 m from the centre of Casey Station
110º32’14”E
Browning
Approximately, 20 km from Casey Station
66º28’20”S;
Peninsula
Low/no human impact
110º32’59”E
ASPA 136
Antarctic Special Protected Area, Active
66º15’08’’S;
Adélie penguin rookery
110º32’15”E
Soil samples collection also was done in Rothera Point-Wright Peninsula, in
Adelaide Island (Fig. 3.2 and Table 3.2). Rothera Research Station (S 67° 34.104'
W068° 06.852') is situated in Antarctic Peninsula, Adelaide Island of East Antarctica.
The site was chosen because it is near to Antarctic Special Protected Area 129 with less
human impact. The site was closed to the sea, with occasional fur seal, crabeater seal or
Adelaide penguin activities. During sampling, the gloves were worn and disinfectant
was used to avoid contamination.
17
Fig. 3.2 Location of Rothera Research Station,
shows the location of Rothera
Research Station. (Map was provided by Dr. Peter Convey from British Antarctic
Survey)
Table 3.2 The sampling sites of Rothera point and isle nearby.
Location
Description
GPS Data
1
Lagoon Island 1
Elephant seal rookery
S 67° 35.689'
W068° 14.495'
2
Lagoon Island 2
Soil and sand from shore
S 67° 35.613'
W068° 14.960'
3
Leonie Island 1
Soil from area with Wedell seal
activity
S 67° 35.607
W068° 20.670'
4
Leonie Island 2
Soil meter away from Antarctic
Skua’s nest
S 67° 35.627'
W068° 20.675'
5
Rothera Point
Soil in the border of Antarctic Special
Protected Area 129
S 67° 34.102'
W068° 06.852'
6
Anchorage
Island 1
Soil under melting snow
S 67° 36.813'
W068° 12.551'
7
Anchorage
Island 2
Soil from rocky mountain
S 67° 36.223'
W068° 12.447'
18
8
Anchorage
Island 3
Yellowish soil under the rock
(without expose to the sun)
S 67° 36.160'
W068° 12.455'
9
Donnelly Island
Rocky soil
S 67° 36.153'
W068° 12.156'
10
Killingbeck
Island
Rocky soil, with Blue-eyed shag
activity
S 67° 35.312
W068° 07.156'
3.4 Enrichment Medium
A basal medium (KG medium) was used to isolate of soil QQ bacteria. The
recipe of the medium in final concentration: (in grams per litre) NaCl, 1.0; KCl, 0.5;
MgCl2 0.4; CaCl2, 0.1; Na2SO4, 0.15; KH2PO4, 5.0; and 2-(N-morpholino)ethanesulfonic acid (MES), 1.0. The pH of this basal medium was adjusted to 5.5 with 1
M NaOH follow by autoclaving. Finally, an appropriate amount of a sterile stock
solution of trace elements (FeCl3, 0.1 g of MnCl2, and 0.46 g of ZnCl2) were added
aseptically to final concentration of 1 mg/L (Chan et al., 2009).
In this study, different AHLs were added to select bacteria that can degrade
various kinds of AHLs. These included N-3-oxo-dodecanoyl-L-homoserine lactone (3oxo-C12-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), and N-heptanoyl-Lhomoserine lactone (C7-HSL),. All AHLs were purchased from Sigma-aldrich or gifts
from Prof. Paul William from University of Nottingham.
3.5 Growth Medium
3.5.1 Luria-Bertani (LB) Media
The ingredient of LB media was modified from Sambrook et al., 1989. The
strength of medium has been reduced to 1/10 to reduce carbon catabolite repression.
19
The media consisted of 0.1% w/v tryptone, 0.05% w/v NaCl, 0.1 % w/v yeast extract, in
1 L of sterile distilled waster (sdH2O). All ingredients were dissolved in sdH2O and
autoclave. 50 mM 3-[N-morpholino] propaesulfonic acid (MOPS) might be added to
adjust to pH 6.8 to avoid lactonolysis which may happen in pH 7.0 and above.
For culturing of biosensor and preparation of competent cells, LB media was
used according to Sambrook’s protocol. The medium consisted of 1.0% w/v tryptone,
0.5% w/v NaCl, 1% w/v yeast extract, in 1 L of sdH2O. All ingredients were dissolved
in distiled water and autoclaved. The biosensors used were mini-Tn5 mutant of
Chromobacterium violaceum (CV026), Escherichia coli [pSB 401], and Escherichia coli
[pSB 1075].
3.5.2 AB Medium
The Agrobacterium tumefaciens NTL4 (pZLR4) was grow in AB medium with
25µg/ml gentamicin as describe in Shaw et al.,1997. The ingredients included:0.4 g
yeast extract, 1.0 mg FeSO4.7H2O, 1.3 g K2HPO4 , 0.4 g NaH2PO4, 0.4 g NH4Cl, 0.12 g
MgSO4.2H2O, 0.06 g KCl , and 4 mg CaCl2.H2O were all dissolved in 400 ml of sdH2O.
3.5.3 SOB medium
SOB medium was used to culture of cloning host (E.coli) before transformation.
SOB medium was prepared as described by Sambrook et al. 1989 and consisted of 2%
w/v tryptone, 0.5% w/v yeast extract, 10 mM NaCl, and 2.5 mM KCl, in 1,000 ml
sdH2O. After sterilization by autoclaving, 5 ml of filter-sterilized (0.22 μm pore size) 2
M MgSO4 was added aseptically to the medium before use.
20
3.5.4 SOC Medium
S.O.C medium was prepared as described by Sambrook et al. 1989 consisted of
0.05% w/v NaCl, 0.36% w/v glucose, 0.5% w/v yeast extract, 0.5% w/v MgSO4.7H2O
and 2.0% w/v tryptone. SOC medium was used to grow E. coli transformed with
plasmids for cloning of 16s rDNA PCR product to increase transformation efficiencies.
3.6 Antibiotics Supplementation
Growth media was supplemented with ampicillin with final concentration of
100µg/ml for E. coli or gentamicin for A.tumefaciens NTL4 (pZLR4) with final
concentration of 30 µg/ml when required. Both the ampicillin and gentamicin stock
solutions were diluted in dH20, filter-sterilized (0.2 µm pore size diameter) and stored at
-20ºC.
3.7 Measurement of Bacterial Growth
Measurements of optical density at 600nm (OD600) were performed by using
Shimadzu UV3600 spectrophotometer. All the bacteria cultures were grown until
OD600=1.0 before harvested for AHLs extraction or AHLs inactivation assay.
3.8 Soil Analysis
Soil samples were processed at Rothera Research Station by drying at 70°C until
constant mass to determine water content for measurements of pH and salinity. Soil pH
was measured in 1:2 (w/v) suspensions of dry soil in distilled water. Salinity, measured
21
as electrical conductivity (µS/cm), was measured in 1:5 (w/v) suspensions of dry soil in
water.
3.9 Detection of N-acyl-homoserine lactone
3.9.1 Extraction of N-acyl-homoserine lactone of Antarctic Bacteria
AHLs were extracted from Antarctic Bacteria by using ethyl-acetate (analytical
grade, Merck). The bacteria were cultured in one tenth strength LB-MOPS at 4°C with
150 rpm until late exponential phase or early stationary phase. The cells were spun
down and the supernatants were mixed with an equal volume of ethyl-acetate. The
mixture was vortexed vigorously for 2 min and then centrifuged at 4400 g for 20 min
(Sartorius stedim model: BBI-8581240). The ethyl acetate layer was transfered into a
new falcon tube and evaporated to dryness under a fume hood. The resultant solid may
comprise AHLs were reconstituted in ethyl acetate (for thin layer chromatography) or
Acetonitril (ACN) (HPLC grade, Fisher Scientific) and were analyzed by LCMS/MS.
3.9.2 Thin Layer Chromatography
Thin layer chromatography (TLC) plates were purchased from Merck. The
protocol to detect AHLs was carried out according to Mclean et al., 1997. Two types of
TLC plate were used to detect AHLs, RP18 plate was used with biosensor CV026 to
detect short chain AHLs, and RP2 was used with biosensor A.tumefaciens NTL4 to
detect long chain AHLs. CV026 can detect a range of short chain AHLs including C4HSL, C5-HSL, C6-HSL, C7-HSL, C8-HSL, 3-oxo-C4-HSL, 3-oxo-C6-HSL and 3-oxoHSL. A.tumefaciencs NTL4, a broad range AHLs biosensor, can detect both short and
22
long chains AHLs. However, A.tumefaciens was not sensitive to certain short chain
AHLs including C5-HSL, C6-HSL and 3-oxo-C6-HSL. The extracted AHLs were
spotted on the TLC plate and the standards were spotted in different lanes. The TLCs
were developed in methanol: water in 60:40 v/v (RP18) and 45:55 v/v (RP2) in sealed
TLC tank, then air-dried and overlaid with 300 ml soft LB agar (seeded with the
biosensor CV026) or with AB agar (seeded with A.tumefaciens NTL4).
3.9.3 Detection of AHL using lux reporter strains
Two bio-reporter strains were used to detect the short chain and long chain
AHLs. These two strains were E. coli [pSB-1075] and E. coli [pSB-401]. With
exogenously supplied AHLs, both strains will produce bioluminescence that can be
detected by a luminometer (Tecan Infinite F500). The various AHL extracts were
placed in the 96 well Elisa plate and dried in the fume hood. Then the bio-reporter strain
which was grown to OD6000.5 was then placed in each well and incubated at 37°C
overnight. The luminometer will take the reading every 15 min. The bioluminescence
produced by the bio-reporter strain was compared with the positive control (synthetic
AHLs).
Table 3.3 Bacterial strains used in this study
Bacterial strain
Description
Source/Reference
Chromabacterium
It is a double mini-Tn5 mutant and acts McClean et al., 1997
violaceum CV026
as a biosensor by producing purple
violacein pigment when short chain
exogenous
AHLs
molecules
are
23
provided.
Erwinia carotovora It produces AHL molecules which can Dr. Chan Kok Gan,
Attn
be detected by using C. violaceum Department
of
CV026 as the biosensor. Thus it was Genetics, University
used as the positive control in quorum of Malaya
sensing test.
Erwinia carotovora It does not produce AHL molecules Dr. Chan Kok Gan,
A20
detectable
by
Chromabacterium Department
of
violaceum CV026. Thus it was used as Genetics, University
the negative control in quorum sensing of Malaya
test.
Agrobacterium
It can detect certain short chain and long Dr. Chan Kok Gan,
tumefaciens
chain AHL molecules. It was used to Department
NTL4(pZLR4)
determine the AHL molecules produced Genetics, University
of
by the bacterial strains which cannot be of Malaya
detected by using the Chromobacterium
violaceum CV026 as the biosensor.
Bacillus cereus
It has a very strong quenching activity Dr. Chan Kok Gan,
towards OC6, C7-HSL and C8-HSL. Department
of
Thus it was used as a positive control for Genetics, University
AHLs inactivation assay.
Escherichia
DH5α
of Malaya
coli It has no quenching activity towards Dr. Chan Kok Gan,
OC6, C7-HSL and C8-HSL. Thus it was Department
of
used as a negative control AHLs Genetics, University
24
inactivation assay. It was also used as of Malaya
competent
cells
for
plasmid
transformation.
3.9.4 Characterisation of AHL using Liquid Chromatography Mass Spectrometry
(LC-MS)
Although AHLs can be detected sensitively by TLC overlay assay using
biosensors (up to submicromole), these bioassays have inherent detection biasness
because they depend on the specificity of a particular reporter strain (Gould et al., 2005).
For example CV026, which produces the purple pigment violacein, will be inhibited by
long chain AHLs (McClean et al., 1997)
Therefore to study all the AHLs produced by bacteria, LC-MS is the more
reliable method. LC-MS detect AHLs based on the chemical properties of AHLs, such
as collisionally induced dissociated ionization product ions, mass/charge ratio of
molecular ions and chromatographic retention properties. This method can be used to
detect and quantify all types of AHLs including N-acyl-, N-hydroxy, and 3-oxoacylhomoserine lactones.
The Agilent RRLC 1200 system was used as the LC delivery system. The
column used in the LC analysis is Agilent ZORBAX Rapid Resolution HT column (1.8
µm, 100 × 2.1mm). The analysis was carried out at 45 ºC with flow rate of 0.4 mL/min,
and the injection volume is 20 µL. Mobile phase A and B were 0.1% formic acid in
water and 0.1% formic acid in acetonitrile, respectively. The gradient profile is
summarized below in Table 2.4.
25
The high resolution MS/MS analysis was performed on an Agilent 6500 Q-TOF
MS/MS system. The MS experiment was performed in the ESI-positive mode. The
probe capillary voltage was set at 3,000 V; desolvation temperature 350 ºC; sheath gas
11 mL/hr; and nebulizer pressure 50 psi. Nitrogen gas was used as the collision gas in
the collisionally induced dissociation (CID) mode for the MS/MS analysis, with
collision energy set at 20 eV. The MS data was analyzed using Agilent MassHunter
software.
Table 3.4 LC gradient
Time (min)
Mobile phase A
Mobile phase B
0.0
70%
30%
5.0
20%
80%
7.0
5%
95%
10.0
5%
95%
11.0
70%
30%
13.0
70%
30%
3.9.5 Synthetic AHL
C7-HSL, 3-oxo-C6-HSL and C8-HSL were obtained from Sigma (USA). C7HSL was dissolved to a concentration of 0.5µg/µl by using absolute ethanol while 3oxo-C6-HSL and C8-HSL were dissolved to a concentration of 1.0 µg/ µl by using
absolute ethanol prior to use.
3.9.6 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal)
X-gal of 100mg/ml was used in the QS test using A. tumefaciens as the biosensor
and its structure is shown in Figure 2.5.
26
3.9.7 Gram staining solutions
Gram staining was performed by using BDTM Gram Stain Kit as per the
manufacturer’s instructions.
3.10 Detection of QQ Activity
3.10.1 Preparation of resting cells
Each bacterial strain was cultured in 8ml of LB broth at 4ºC until OD600 1.0 at
180 rounds per minute (rpm). E. coli as the negative control was cultured at 37 ºC.
Culture (1.5ml) was harvested at 12,500 g for 4 min. The supernatant was discarded and
the pellet was resuspended in 300 µl phosphate buffer saline (PBS) (100 mM, pH 6.5).
Then the cell suspension was washed twice by using 300 µl PBS and finally
resuspended in 100 µl of the same buffer.
3.10.2 AHL inactivation assay
A total of 5 µl of C7-HSL (0.5 µg/µl) was evaporated to dryness in sterile 1.5 ml
tubes. Then, 100 µl of resting cell suspension (Section 3.9.1) was used to rehydrate C7HSL providing C7-HSL a final concentration of 0.025 µg/µl. The suspensions were then
incubated at 28ºC with shaking at 220 rpm. Aliquots of 25 µl cell suspension was
withdrawn at 0 h, 4 h, 9 h and 24 h prior to heat inactivation at 95ºC for 5 min. Then, 10
µl of inactivated cell suspension at different intervals were used in the overlaid with
CV026.For AHL inactivation assay with 3-oxo-C6-HSL and C8-HSL, the above
procedures were repeated except that 5 µl of 3-oxo-C6-HSL (0.10 µg/µl) or C8-HSL
(1.0µg/ µl) were used.
27
3.10.3 Rapid Resolution Liquid Chromatography (RRLC)
Rapid resolution liquid chromatography (RRLC) is the most reliable method to
detect QQ activity. AHL was separated and quantified by using RRLC (Agilent).
Reverse-phase RRLC analysis of AHL was carried out as reported (Swift et al., 1996)
with slight modification. Analysis was performed using an Agilent Technologies 1200
Series Rapid Resolution LC system (Agilent Technologies, Germany) equipped with a
vacuum degasser, a binary pump SL, an autosampler, and a diode-array detector (DAD).
Ten microliters of extracted AHL from AHL inactivation assay were applied onto an
analytical C18 reverse-phase column (Agilent ZORBAX Eclipse® XDB-C18, 4.6
mm×50 mm, 1.8 µm particle size). RRLC was run on isocratic profile of acetonitrilewater (35:65, v/v, for short-chain AHL; 60:40, v/v, for long-chain AHL) for 3 min at a
constant flow rate of 0.7 ml/min and spectrum monitored at 210 nm. Data collection and
analysis were performed using Agilent Chemstation (version B.04.01). Both the
retention time and spectral properties were compared to a series of synthetic AHL
standards obtained from Sigma-Aldrich®. The percentage of AHL inactivated and the
specific activity were determined by estimating the amount of AHL (by comparison of
the reduction in peak areas for a given retention time) with respect to AHL solutions of
known concentration. Then, 100 µg AHLs in absolute ethanol were dispensed into two
1.5 ml sterile tubes where the solvent was evaporated to dryness. The tubes were then
filled with 100 µl of bacteria culture isolate from Antarctic soil, rehydrating the AHLs
to a final concentration of 1 µg/µl. The resting cell suspensions were incubated at 4°C
with shaking (150 rpm). Residual AHLs reacted were extracted at 0 h and 24 h by using
ethyl acetate and evaporate do dryness. The dried AHL was then dissolved in
acetonitrile (HPLC grade).
28
3.11 16S rDNA analysis and phylogenetic study
The genomic DNA of Antarctic bacteria were extracted using Qiagen QIAamp
DNA Mini Kit. The 16S ribosomal DNA (rDNA) universal primers 27F (Weisburg et
al., 1991) and 1525R (Dewhirst et al., 1999) were then used for 16S rDNA PCR with
bacteria isolate genomic DNA as the template. PCR amplification and purification of
16S rDNA products were carried out and the PCR products were ligated into pGEM-T
by using the pGEM-T Vector System (Promega, USA). DNA sequencing was
performed by routine automated methods in which standard M13 forward and reverse
primers and primers previously designed to anneal to internal target regions of the 16S
rDNA of most bacteria were used (Lane et al., 1985). The nucleotide sequence of a
PCR-amplified fragment of the 16S rDNA of the isolates were determined as described
previously (Chan et al., 2009) and we used MEGA version 4 (Tamura et al., 2007) to
perform phylogenetic and molecular evolutionary relationship analyses. A phylogenetic
tree was generated by using the neighbour-joining algorithm and rooted with an
appropriate outgroup to show the direction of evolution. Bootstrap analyses for 1,000
re-samplings were performed to provide confident estimates for tree topology.
Before sending the 16s rDNA of bacteria isolate to sequencing service, bacteria
that look alike morphologically and functionally (similar QS and QQ activities), colony
PCR and RFLP analysis were used to distinguish between different bacterial species to
avoid using the same bacterial species in this study.
3.11.1Genomic DNA extraction
Genomic DNA of each bacterial strain was extracted by using QIAamp® DNA
Mini Kit and the protocols in the commercial kit were followed. Agarose gel
electrophoresis was done to confirm the genomic DNA extracted.
29
3.11.2 PCR purification and Gel extraction
PCR product purification and gel extraction was carried out by using QIAquick
gel extraction kit.
3.11.3 Primers
The primers that were used to amplify 16S rDNA gene and this study are listed
in Table 3.5.
Table 3.5 Primers used in this study.
Primers
Sequences
Sources
16S rDNA
5’-AGAGTTTGATCMTGGCTCAG-3’
Operon
5’-AAGGAGGTGWTCCARCC-3’
Operon
5’-ACTCCTACGGGAGGCAGCAG-3’
Operon
5’-GTGCCAGCAGCCGCGGTAA-3’
Operon
forward primer
27F
16S rDNA
reverse primer
1525R
16S rDNA
forward primer
338F
16S rDNA
forward primer
30
515F
5’-AGGATTAGATACCCTGGTAGTCCA-3’
Operon
5’-GAGGAAGGTGGGGATGACGT-3’
Operon
SP6
5’-TTCTATAGTGTCACCTAAAT-3’
Operon
T7
5’-TAATACGACTCACTATAGGG-3’
Operon
16S rDNA
forward primer
783F
16S rDNA
forward primer
1174F
Legend :
M = A+C, W = A+T, R = A+G
3.11.4 PCR master mix preparation
The PCR kit (Intron) was thawed on ice. The volume of components needed to
prepare the required number of reactions was calculated as below.
31
Table 3.6 Volume and components for PCR master mix preparation
Component
Volume/Reaction(µL)
10× PCR Buffer
2.0
25× dNTP Mix(100 mM)
0.8
16S rDNA primer
2.0
Taq DNA polymerase
0.5
Ultra pure water (MiliQ)
9.7
Total per Reaction
15.0
The master mix was thawed on ice and well mixed. The mixtures briefly
centrifuged to spin down the contents. The tubes were placed on ice until ready to be
loaded into the thermal cycler (Applied Biosystem, USA).
3.11.5 Agarose gel and electrophoresis
Briefly, 1% w/v of the agarose gel in TBE buffer was used. Ethidium bromide
(0.5 ug/ml) was added to visualise the DNA bands under UV.
3.11.6 DNA size reference markers
iNtRON 100bp Ladder Molecular Weight DNA Marker and Vivantis 1kb DNA
Ladder were used.
32
3.11.7 Preparation of Chemically Competent Cells
Competent cell (E. coli DH5α) was prepared to clone the 16s rDNA. Briefly,
5ml of LB broth was inoculated with a single colony of selected E. coli DH5α and
incubated overnight at 37°C, in 250 rpm. Then, 50 ml of LB broth then added by
inoculated with the overnight bacteria culture above for at 37°C (250 rpm) until OD600
0.5. Culture was placed on ice in sterile, prechilled Corex tubes for 10 min, follow by
centrifuged for 7 min at 2500rpm (4°C). The pellet was then resuspended in ice-cold
10ml CaCl2 solution (60mM CaCl2, 15% v/v glycerol, 10mM PIPES, pH 7.0) .The cells
was centrifuged for 5 min at 2500rpm (4oC) and then resuspended the pellet in 10ml
CaCl2 solution. The cells were incubated on ice for 30 min and then centrifuged for 5
min(2500rpm, 4°C) and resuspended the cells in 2ml CaCl2 solution. Aliquot of cells
(100ml) was rapid freezed on dry ice and stored at -70oC.
3.11.8 Ligation, Transformation and Cloning
The following solutions (Table 3.7) were added and incubated overnight.
Table 3.7 Volume and component for ligation reaction
Reagent
Volume
2 × rapid ligation buffer
2.5 µl
pGEMT®-T easy vector
0.5 µl
T4 DNA ligase
0.5 µl
Purified PCR product
1.5 µl
33
The tubes containing purified PCR product were briefly centrifuged to collect
the PCR product. The 2× rapid ligation buffer was vortexed to mix well before use.
The tubes containing the ligation reaction were briefly centrifuged. Competent
cells were thawed on ice for about 5 min. Then, 2.0 µl of each ligation reaction were
dispensed into sterile 1.5 ml tubes on ice. Competent cells were added into the tubes
containing the ligation reaction by using chilled micropipette tips. The tubes were then
flicked gently to mix well and placed on ice for 20 min. The cells were then heat
shocked at 42ºC for 45 s, followed by placing on ice for 2 min. After which, 950µl of
warm SOC medium were added into the competent cells. The tubes were incubated at
37ºC for 1 h 30 min with regular shaking of 220 rpm. Then 200 µl of the cell suspension
were spread onto the LB agar plate with ampicilin. The plates were then incubated at
37ºC for overnight.
Colonies grown on the LB agar with ampicilin were picked and PCR was
performed to check for trasformant. Universal primers SP6 and T7 were used. The PCR
conditions were as follow: 1 cycle of initial denaturation at 94 ºC for 5 min; 25 cycles of
denaturation at 94 ºC for 30 s; annealing at 58 ºC for 30 s and extension at 72 ºC for 1
min and 30 s; and 1 cycle of final DNA extension at 72 ºC for 7 min. On agarose gel
electrophoresis, amplicon of about 1.7kb indicates that the colony selected was a
transformant.
3.11.9 Plasmid extraction
Single colony of transformant was inoculated in LB broth with ampicillin. The
culture was then incubated and shaken at 37 ºC overnight. QIAprep Spin Mini Kit was
34
used to extract the plasmid and it is performed according to manufacturer’s instruction.
The plasmid DNA was kept at -20ºC.
3.11.10 Sequencing Analysis
A total of 20 µl of plasmid DNA (100 ng/µl) for each bacterial strain were sent
to MACROGEN for sequencing. Four forward primers, 338F, 515F, 783F and 1174F
were used in primer walk-up sequencing to obtain the complete sequence of 16S rDNA.
Nucleotide sequences were aligned by using MegAlign (DNASTAR) and analyzed by
using SeqEdit (DNASTAR). The nucleotide sequences for the four primers were
aligned and trimmed to obtain a segment of 16S rDNA for the identification of the
species. Standard nucleotide-nucleotide BLAST program which was available in the
NCBI website (http://www.ncbi.nlm.nih.gov) was used to compare the nucleotide
sequences obtained with those stored in the GenBank database.
3.11.11 Phylogenetic Analysis
Phylogenetic analysis was done by using the Molecular Evolutionary genetic
Analysis (MEGA) version 5 downloaded from http://www.megasoftware.net. (Tamura
et al., 1997). First the 16S rDNA sequences obtained which have been exported to the
FASTA file type were aligned by using the MEGA 4.1. Then a phylogenetic tree could
be built by using Bookstrap test of phylogenetic and neighbor-joining method. The
model used was nucleotide maximum composite likelihood (MCL) to estimate the
evolutionary distances between all sequences at one time. This MCL model is likely to
have reduced error and thus the evolutionary distance could be estimated more
accurately by the distance-based method.
35
CHAPTER 4
RESULTS
4.1 Isolation of Novel QQ Bacteria
Strain R3.7 was enriched by using KG medium supplemented with C7-HSL (Nheptanoyl-L-Homoserine lactone) inoculated with soil sample from Rothera Research
Staion (ASPA 129) at 4°C. This strain is Gram negative bacteria. The growth of strain
R3.7 was maintained on LB agar. The colony morphology for this strain is nonpigmented, circular and smooth. The cell morphology under microscope is non-motile,
coccobacilli, some of the cells occurring in group. After 168 h and three transfers in
fresh KG medium, pure colonies were obtained. The 16S rDNA gene analysis of isolate
R3.7 suggested that this isolate is clustered to Psychrobacter sp. (Fig. 4.1).
Strain L10.15 was isolated from soil sample collected from elephant seal
rookery, Lagoon Island (S 67° 35.689', W068° 14.495') (Table 3.2). Strain L10.15 was
also enriched by using KG medium supplemented with C7-HSL in 4°C. The strain is
Gram positive bacteria. Strain L10.15 was growth on LB agar. The colonies
morphology for this strain is orange-pigmented, opaque, smooth, convex and circular.
The cell morphology under microscope is motile, coccoid and occurring in pair or group.
After 168 h and 3 transfers in fresh KG medium, pure colonies were obtained. The 16S
rDNA gene analysis of isolate L10.15 suggests that this isolates clustered closely to
Planococcus sp. (Fig. 4.2).
Strain Ln5B.12 was isolated in Leonie Island (S 67° 35.607' W068° 20.670')
whereby the soil was collected from shore with wedell seal activity. Strain Ln5B.12 was
also enriched with KG medium supplemented with C7-HSL in 4°C. The strain is Gram
negative bacteria. Strain Ln5B.12 was growth on LB agar. The colonies morphology for
36
this strain is dark yellow-pigmented, circular and smooth. The cell morphology for
strain Ln5B.12 is non-motile and rod shape under microscope. After 168 h and 3
transfers in fresh KG medium, pure colonies were obtained. The 16S rDNA gene
analysis of isolate Ln5B.12 suggests that this isolate clustered closely to
Flavobacterium sp. (Fig. 4.3).
4.1.1 Phylogenetic Analysis of Novel QQ Bacteria
Web-based similarity searches against sequences in the GenBank suggested that
strain R3.7 is closed to Psychrobacter cryohalolentis (99% sequence identity with
P.cryohalolentis strain KOPRI 22219, gene accession number:
EU090718).
Phylogenetic analysis supported that the closest species for strain R3.7 is strain of
P.cryohalolentis KOPRI 22219 (Fig. 4.1).
Fig.4.1: Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of strain R3.7 with closely
related taxa. Bar represents evolutionary distance as 0.01 change per nucleotide position.
Bootstrap values (%) over 50% from 1,000 replications are shown. Moraxella
catarrhalis ATCC 25238 (gene accession number: AY123721) was used as outgroup.
37
Web-based similarity searches against sequences in the GenBank suggested that
strain L10.15 closest to Planococcus antarcticus (98% sequence identity with
Planococcus antarcticus strain R-36948, gene accession number: FR691465). (Fig. 4.2).
Fig.4.2: Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of strain L10.15 with closely
related taxa. Bar represents evolutionary distance as 0.005 change per nucleotide
position. Bootstrap values (%) over 50% from 1,000 replications are shown.
Sporosarcina macmurdoensis (gene accession number: AJ514408) was used as
outgroup.
38
Web-based similarity searches against sequences in the GenBank suggested that
strain Ln 5B.12 closest to Flavobacterium hercynium (97% sequence identity with
Flavobacterium hercynium strain WB 4.2-78, gene accession number: AM177627 ) (Fig.
4.3).
Fig.4.3: Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of strain L10.15 with closely
related taxa. Bar represents evolutionary distance as 0.01 change per nucleotide position.
Bootstrap values (%) over 50% from 1,000 replications are shown. Myroides odoratus
(Gene accession numbe :M58777) was used as outgroup.
39
4.1.2 Degradation of Various AHLs by Novel QQ Bacteria
Psychrobacter sp. R3.7 was found to be able to inactivate all the N-acyl
homoserine lactones tested. The range of AHLs tested was from C4-C13 and this strain
is able to degrade AHL with or without substitution group (hydroxy and oxo). Strain
R3.7 was also found to produce a cold adapted AHL lactonase-type enzyme (Figure 4.64.9).
The QQ ability of strain L10.15 was limited to AHLs with acyl side chain up to
C10. This bacteria showed a preference to degrade short chain AHLs. However, within
the range of C4-C10 AHLs, this bacterial was able to degrade AHLs with or without
substitution group (hydroxy and oxo). This bacteria was also found to be able to
degrade another type of signaling molecule (HHQ) which is produced by Pseudomonas
spp. in Antarctica.
Flavobacterium sp. strain Ln5B.12 was observed to quench short chain AHLs
range from C4-C8 HSL. This bacteria was also found to possess QS activity as well.
The positive result for biosensor E.coli [pSB 1075] showed that this bacteria was
producing long chain AHLs. However, the mechanism of degradation of short chain
AHLs remains unknown. The AHLs were found failed to recover after acidified with
HCl. This indicates that inactivation of AHLs by this bacteria is not via lactonase-type
of enzyme.
40
4.1.3 RRLC Analysis of Degradation of AHLs by Psychrobacter sp. strain R3.7
RRLC analysis of degradation of AHLs by Psychrobacter sp. strain R3.7.
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-08 11-51-42\2BA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-08 11-51-42\2AB-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-08 11-51-42\2AC-1501.D)
mAU
200
0h
175
150
125
24 h
100
75
50
25
48 h
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Fig.4.4.1a RRLC analysis of synthetic C4-HSL (100µg) incubated with Psychrobacter
sp. strain R3.7 at 0-, 24-, and 48-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-08 11-51-42\2BA-0801.D )
mAU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
min
0.5
1
1.5
min
Figure 4.4.1b (left) and 4.4.1c (right) RRLC analysis of synthetic C4-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C4-HSL at 0-h
Chromatogram of C4-HSL at 24-h
Chromatogram of C4-HSL at 48-h
41
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \2 A B -2 9 0 1 .D )
0 3 -2 5 -5 1 \2 B B -3 0 0 1 .D )
0 3 -2 5 -5 1 \2 C B -3 1 0 1 .D )
0 3 -2 5 -5 1 \2 D B -3 2 0 1 .D )
mAU
200
175
150
48 h & 72 h
125
100
75
50
25
0
0 .5
1
m in
1 .5
Fig.4.4.2a RRLC analysis of synthetic C6-HSL (100 µg) with Psychrobacter sp. strain
R3.7, at 0-, 24-, 48- and 72- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2001.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2401.D )
mAU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
min
0.5
1
1.5
min
Figure 4.4.2b (left) and 4.4.2c (right) RRLC analysis of synthetic C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram C6-HSL at 0-h
Chromatogram C6-HSL at 24-h
Chromatogram C6-HSL at 48-h
Chromatogram C6-HSL at 72-h
42
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \2 A C -4 5 0 1 .D )
0 3 -2 5 -5 1 \2 B C -4 6 0 1 .D )
0 3 -2 5 -5 1 \2 C C -4 7 0 1 .D )
0 3 -2 5 -5 1 \2 D C -4 8 0 1 .D )
mAU
350
0h
300
24 h
250
48 h
200
72 h
150
100
50
0
0 .5
1
m in
1 .5
Fig.4.4.3a RRLC analysis of synthetic 3-Oxo-C6-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48- and 72-h at 10°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -4001.D )
2011-08-09 03-25-51\2AC -3301.D )
2011-08-09 03-25-51\2AC -3401.D )
2011-08-09 03-25-51\2AC -3501.D )
2011-08-09 03-25-51\2AC -3601.D )
m AU
mAU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.4.3b(left) and 4.4.3c (right) RRLC analysis of synthetic 3-oxo-C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C6-HSL at 0-h
Chromatogram of 3-Oxo-C6-HSL at 24-h
Chromatogram of 3-Oxo-C6-HSL at 48-h
Chromatogram of 3-Oxo-C6-HSL at 72-h
43
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \2 A D -6 1 0 1 .D )
0 3 -2 5 -5 1 \2 B D -6 2 0 1 .D )
0 3 -2 5 -5 1 \2 C D -6 3 0 1 .D )
0 3 -2 5 -5 1 \2 D D -6 4 0 1 .D )
mAU
80
60
0h
40
20
24 h
0
48 h
72 h
0 .5
1
m in
1 .5
Fig.4.4.4a RRLC analysis of synthetic 3-Hydroxy-C6-HSL (100µg) with Psychrobacter
sp. strain R3.7, at 0-, 24-, 48 and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -4901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5001.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5201.D )
mAU
mAU
80
80
60
60
40
40
20
20
0
0
0.5
1
1.5
min
0.5
1
1.5
min
Fig.4.4.4b (left) and 4.4.4c (right) RRLC analysis of synthetic 3-hydroxy-C6-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Hydroxy-C6-HSL at 0-h
Chromatogram of 3-Hydroxy-C6-HSL at 24-h
Chromatogram of 3-Hydroxy-C6-HSL at 48-h
Chromatogram of 3-Hydroxy-C6-HSL at 72-h
44
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2AA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2BA-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2CA-1501.D)
mAU
0h
160
140
120
100
24 h
80
60
40
20
48 h
0
0.25
0.5
0.75
1
1.25
1.5
1.75
min
Fig.4.4.5a RRLC analysis of synthetic C7-HSL (100µg) with Psychrobacter sp. strain
R3.7, at 0-, 24-, and 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0401.D )
mAU
175
mAU
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
min
0.5
1
1.5
min
Fig.4.4.6b (left) and 4.4.6c (right) RRLC analysis of synthetic C7-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C7-HSL at 0-h
Chromatogram of C7-HSL at 24-h
Chromatogram of C7-HSL at 48-h
45
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \2 A B -2 9 0 1 .D )
1 7 -0 6 -0 3 \2 B B -3 0 0 1 .D )
1 7 -0 6 -0 3 \2 C B -3 1 0 1 .D )
1 7 -0 6 -0 3 \2 D B -3 2 0 1 .D )
mAU
30
0h
20
24 h
10
48 h
0
72 h
-1 0
0 .5
1
m in
1 .5
Fig.4.4.6a RRLC analysis of synthetic C8-HSL (100µg) with Psychrobacter sp. strain
R3.7, at 0-, 24-, 48- and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-2101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-2201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-2301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-2401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-1701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-1801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-1901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AB-2001.D )
mAU
mAU
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
min
0.5
1
1.5
min
Fig.4.4.6b (left) and 4.4.6c (right) RRLC analysis of synthetic C8-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatgram of C8-HSL at 0-h
Chromatgram of C8-HSL at 24-h
Chromatgram of C8-HSL at 48-h
Chromatgram of C8-HSL at 72-h
46
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \2 A C -4 5 0 1 .D )
1 7 -0 6 -0 3 \2 B C -4 6 0 1 .D )
1 7 -0 6 -0 3 \2 C C -4 7 0 1 .D )
1 7 -0 6 -0 3 \2 D C -4 8 0 1 .D )
mAU
0h
250
200
150
24 h
100
50
48 h
72 h
0
0 .5
1
m in
1 .5
Fig.4.4.7a RRLC analysis of synthetic 3-Oxo-C8-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48-h and 72- h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -4001.D )
2011-08-09 17-06-03\2A C -3301.D )
2011-08-09 17-06-03\2A C -3401.D )
2011-08-09 17-06-03\2A C -3501.D )
2011-08-09 17-06-03\2A C -3601.D )
m AU
mAU
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.4.7b (left) and 4.4.7c (right) RRLC analysis of synthetic 3-oxo-C8-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C8-HSL at 0-h
Chromatogram of 3-Oxo-C8-HSL at 24-h
Chromatogram of 3-Oxo-C8-HSL at 48-h
Chromatogram of 3-Oxo-C8-HSL at 72-h
47
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2AD-6101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2BD-6201.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\2CD-6301.D)
mA
0h
3
3
24 h
2
2
1
1
5
48 h
0
0.2
0.
0.7
1
72 h
1.2
1.
mi
1.7
Fig.4.4.8a RRLC analysis of synthetic 3-Hydroxy-C8-HSL (100µg) with Psychrobacter
sp. strain R3.7, at 0-, 24-, and 48- h and 72-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5601.D )
2011-08-09 17-06-03\2A D -4901.D )
2011-08-09 17-06-03\2A D -5001.D )
2011-08-09 17-06-03\2A D -5101.D )
2011-08-09 17-06-03\2A D -5201.D )
m AU
mAU
40
40
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.4.8b (left) and 4.4.8c (right) RRLC analysis of synthetic 3-Hydroxy-C8-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h and 72-h at 4°C.
Legends:
Chromatogram of 3-Hydroxy-C8-HSL at 0-h
Chromatogram of 3-Hydroxy-C8-HSL at 24-h
Chromatogram of 3-Hydroxy-C8-HSL at 48-h
Chromatogram of 3-Hydroxy-C8-HSL at 72-h
48
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 03-26-01\2AA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 03-26-01\2BA-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 03-26-01\2CA-1501.D)
mAU
0h
70
24 h
60
48 h
50
40
30
20
10
0
0.5
1
1.5
2
min
2.5
Fig.4.4.9a RRLC analysis of synthetic C9-HSL (100µg) with Psychrobacter sp. strain
R3.7, at 0-, 24-, 48-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
2011-08-11 03-26-01\2A A -0101.D )
2011-08-11 03-26-01\2A A -0201.D )
2011-08-11 03-26-01\2A A -0301.D )
2011-08-11 03-26-01\2A A -0401.D )
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
m AU
m AU
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2011-08-11 03-26-01\2A A -0501.D )
2011-08-11 03-26-01\2A A -0601.D )
2011-08-11 03-26-01\2A A -0701.D )
2011-08-11 03-26-01\2A A -0801.D )
0
0.5
1
1.5
2
2.5
m in
0.5
1
1.5
2
m in
2.5
Fig. 4.4.9b (left) and 4.4.9c (right) RRLC analysis of synthetic C9-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h and
72-h at 4°C.
Legends:
Chromatogram of C9-HSL at 0-h
Chromatogram of C9-HSL at 24-h
Chromatogram of C9-HSL at 48-h
49
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
0 3 -2 6 -0 1 \2 A B -3 7 0 1 .D )
0 3 -2 6 -0 1 \2 B B -3 8 0 1 .D )
0 3 -2 6 -0 1 \2 C B -3 9 0 1 .D )
0 3 -2 6 -0 1 \2 D B -4 0 0 1 .D )
mAU
80
0h
70
60
24 h
50
40
48 h
30
72 h
20
10
0
1
2
m in
3
Fig.4.4.10a RRLC analysis of synthetic C10-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48- and 72-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 03-26-01\2A B -2501.D )
2011-08-11 03-26-01\2A B -2601.D )
2011-08-11 03-26-01\2A B -2701.D )
2011-08-11 03-26-01\2A B -2801.D )
m AU
m AU
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
2011-08-11 03-26-01\2AB-2901.D )
2011-08-11 03-26-01\2AB-3001.D )
2011-08-11 03-26-01\2AB-3101.D )
2011-08-11 03-26-01\2AB-3201.D )
0
0
1
2
3
m in
1
2
3
m in
Fig.4.4.10b (left) and 4.4.10c (right) RRLC analysis of synthetic C10-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h and
72-h at 4°C.
Legends:
Chromatogram of C10-HSL at 0-h
Chromatogram of C10-HSL at 24-h
Chromatogram of C10-HSL at 48-h
Chromatogram of C10-HSL at 72-h
50
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
1 1 -1 1 -1 0 \2 A A -1 3 0 1 .D )
1 1 -1 1 -1 0 \2 B A -1 4 0 1 .D )
1 1 -1 1 -1 0 \2 B A -1 5 0 1 .D )
1 1 -1 1 -1 0 \2 D A -1 6 0 1 .D )
mAU
0h
35
24 h
30
48 h
25
20
72 h
15
10
5
0
-5
0 .5
1
m in
1 .5
Fig.4.4.11a RRLC analysis of synthetic C11-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48- and 72- h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AA-0101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AA-0201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AA-0401.D )
2011-08-11 11-11-10\2A A -0101.D )
2011-08-11 11-11-10\2A A -0201.D )
2011-08-11 11-11-10\2A A -0301.D )
2011-08-11 11-11-10\2A A -0401.D )
m AU
mAU
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
0
-5
-5
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.4.11b (left) and 4.4.11c (right) RRLC analysis of synthetic C11-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h and
72-h at 4°C.
Legends:
Chromatogram of C11-HSL at 0-h
Chromatogram of C11-HSL at 24-h
Chromatogram of C11-HSL at 48-h
Chromatogram of C11-HSL at 72-h
51
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
1 1 -1 1 -1 0 \2 A B -3 7 0 1 .D )
1 1 -1 1 -1 0 \2 B B -3 8 0 1 .D )
1 1 -1 1 -1 0 \2 C B -3 9 0 1 .D )
1 1 -1 1 -1 0 \2 D B -4 0 0 1 .D )
mAU
80
70
60
50
0h
40
30
24 h
20
48 h
10
72 h
0
0 .5
1
m in
1 .5
Fig.4.4.12a RRLC analysis of synthetic C12-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48- and 72-h at 4°C.
DAD1
DAD1
DAD1
DAD1
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
2011-08-11
2011-08-11
2011-08-11
2011-08-11
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
11-11-10\2A B -2501.D )
11-11-10\2A B -2601.D )
11-11-10\2A B -2701.D )
11-11-10\2A B -2801.D )
mAU
m AU
50
50
40
40
30
30
20
20
10
10
0
0
0.5
1
1.5
m in
0.5
2011-08-11 11-11-10\2AB-2901.D )
2011-08-11 11-11-10\2AB-3001.D )
2011-08-11 11-11-10\2AB-3101.D )
2011-08-11 11-11-10\2AB-3201.D )
1
1.5
m in
Fig.4.4.12b (left) and 4.4.12c (right) RRLC analysis of synthetic C12-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h and
72-h at 4°C.
Legends:
Chromatogram of C12-HSL at 0-h
Chromatogram of C12-HSL at 24-h
Chromatogram of C12-HSL at 48-h
Chromatogram of C12-HSL at 72-h
52
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
1 1 -1 1 -1 0 \2 A C -6 1 0 1 .D )
1 1 -1 1 -1 0 \2 B C -6 2 0 1 .D )
1 1 -1 1 -1 0 \2 C C -6 3 0 1 .D )
1 1 -1 1 -1 0 \2 D C -6 4 0 1 .D )
mAU
200
175
150
125
100
75
50
25
0
0 .5
1
m in
1 .5
Fig.4.4.13a RRLC analysis of synthetic 3-Oxo-C12-HSL(100µg) with Psychrobacter
sp. strain R3.7, at 0-, 24-, 48-and 72- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 11-11-10\2AC -4901.D )
2011-08-11 11-11-10\2AC -5001.D )
2011-08-11 11-11-10\2AC -5101.D )
2011-08-11 11-11-10\2AC -5201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
m AU
200
mAU
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
2011-08-11 11-11-10\2AC -5301.D )
2011-08-11 11-11-10\2AC -5401.D )
2011-08-11 11-11-10\2AC -5501.D )
2011-08-11 11-11-10\2AC -5601.D )
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.4.13b (left) and 4.4.13c (right) RRLC analysis of synthetic 3-oxo-C12-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h and 72-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C12-HSL at 0-h
Chromatogram of 3-Oxo-C12-HSL at 24-h
Chromatogram of 3-Oxo-C12-HSL at 48-h
Chromatogram of 3-Oxo-C12-HSL at 72-h
53
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 2
2 0 1 1 -0 8 -1 2
2 0 1 1 -0 8 -1 2
2 0 1 1 -0 8 -1 2
0 1 -1 6 -2 6 \2 A A -1 3 0 1 .D )
0 1 -1 6 -2 6 \2 B A -1 4 0 1 .D )
0 1 -1 6 -2 6 \2 B A -1 5 0 1 .D )
0 1 -1 6 -2 6 \2 D A -1 6 0 1 .D )
mAU
80
0h
70
60
24 h
50
40
48 h
30
20
10
0
0 .2
0 .4
0 .6
0 .8
1
1 .2
m in
1 .4
Fig.4.4.14a RRLC analysis of synthetic C13-HSL (100µg) with Psychrobacter sp.
strain R3.7, at 0-, 24-, 48- h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-12 01-16-26\2A A -0101.D )
2011-08-12 01-16-26\2A A -0201.D )
2011-08-12 01-16-26\2A A -0301.D )
2011-08-12 01-16-26\2A A -0401.D )
m AU
mAU
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
2011-08-12 01-16-26\2AA-0501.D )
2011-08-12 01-16-26\2AA-0601.D )
2011-08-12 01-16-26\2AA-0701.D )
2011-08-12 01-16-26\2AA-0801.D )
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
m in
0.2
0.4
0.6
0.8
1
1.2
1.4
min
Fig.4.4.14b (left) and 4.4.14c (right) RRLC analysis of synthetic C13-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C13-HSL at 0-h
Chromatogram of C13-HSL at 24-h
Chromatogram of C13-HSL at 48-h
54
4.1.5 Quantitative Analysis of AHL Degradation by Psychrobacter sp. strain R3.7
Using RRLC analysis, it was determined that Psychrobacter sp. strain R3.7 is
able to inactivate various AHLs.
Table 4.1 Estimation of activity by Psychrobacter sp. strain R3.7 of various AHLs
Types of AHL
E.A*
C4-HSL
31.78
C6-HSL
3.26
3-oxo-C6-HSL
9.09
3-hydroxy-C6-HSL
14.15
C7-HSL
32.27
C8-HSL
32.41
3-oxo-C8-HSL
18.54
3-hydroxy-C8-HSL
29.71
C9-HSL
13.67
C10-HSL
14.60
C11-HSL
17.47
C12-HSL
11.37
3-oxo-C12-HSL
13.08
C13-HSL
15.58
*EA, estimated activity, expressed as ng AHL degraded h-1(105CFU/ml)-1 at 4°C
55
Fig. 4.5 (a) shows the representative graphs to show the degradation of 3-hydroxy-C6HSL by Psychrobacter sp. strain R3.7
Fig. 4.5 (b) shows the representative graphs to show the degradation of 3-oxo-C8-HSL
by Psychrobacter sp. strain R3.7
56
Fig. 4.5 (c) shows the representative graphs to show the degradation of C10-HSL by
Psychrobacter sp. strain R3.7
Fig. 4.5 (d) shows the representative graphs to show the degradation of 3-oxo-C12-HSL
by Psychrobacter sp. strain R3.7
57
Figures 4.5(a), 4.5(b), 4.5 (c) and 4.5(d) are the representative graphs to show
the degradation of AHLs from different group ( short chain AHL, long chain AHL, with
or without substitution group) by Psychrobacter sp. strain R3.7. These graphs show the
amount of AHL for AHL inactivation assay from 0-72 h. PBS buffer (100mM, pH 6.5)
and E. coli DH5α were used as negative controls for the assay.
From the quantitative data (Table 4.1), Psychrobacter sp. strain R3.7 was most
effectively in degrading C8-HSL and 3-hydroxy-C8-HSL. Strain R3.7 could degrade all
AHLs tested but not 3-heptyl-4-quinolone (HHQ) (data not shown).
4.1.6 Determination of AHL Degradation via Lactonase Activity by Psychrobacter
sp. strain R3.7
To determine the AHLs degradation mechanism, strain R3.7 was tested for
lactonase activity, the reacted AHLs showed significant recovery after acidification for
24 h with 100 mM HCl (pH 2.0). For C4-HSL, the amount of AHL was recovered up to
30% (Fig. 4.5 (b)). The recyclization of the lactone ring was also observed in odd
number side chain namely C7-HSL (46% recovery) (Fig.4.6 (b)), 3-hydroxy-C8-HSL
(70% recovery) (Fig.4.7 (b)) and long chain AHL C13-HSL (10% recovery) (Fig.4.8
(b)).
58
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-08 11-51-42\2BA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-08 11-51-42\2AB-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-08 11-51-42\2AC-1501.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-08 11-51-42\2AD-1601.D)
mAU
0h
200
175
150
24 h
125
100
75
acidification
50
25
48 h
0
0.5
1
min
1.5
Fig 4.6 (a) Chromatogram of post- Fig 4.6 (b) Graph of concentration of C4acidification recovery of C4-HSL by strain HSL
R3.7
shows
the
post-acidification
recovery of C4-HSL by strain R3.7
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2AA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2BA-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2CA-1501.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2DA-1601.D)
mAU
175
0h
150
125
100
75
24 h
acidification
50
25
0
48 h
0.5
1
1.5
min
Fig 4.7 (a) Chromatogram of post- Fig 4.7 (b) Graph of concentration of C7acidification recovery of C7-HSL by strain HSL
R3.7.
shows
the
post-acidification
recovery of C4-HSL by strain R3.7.
59
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2AD-6101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2BD-6201.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2CD-6301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 17-06-03\2DD-6401.D)
mAU
40
0h
30
24 h
acidification
20
10
48 h
0
-10
0.5
1
min
1.5
Fig 4.8 (a) Chromatogram of post- Fig 4.8 (b) Graph of concentration of 3acidification recovery of 3-hydroxy-C8- hydroxy-C8-HSL
HSL by strain R3.7.
shows
the
post-
acidification recovery of C4-HSL by strain
R3.7.
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-12 01-16-26\2AA-1301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-12 01-16-26\2BA-1401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-12 01-16-26\2BA-1501.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\TEST 2011-08-12 01-16-26\2DA-1601.D)
mAU
0h
80
70
24 h
60
acidification
50
40
48 h
30
20
10
0
0.2
Fig.
4.9
0.4
(a)
0.6
0.8
1
1.2
Chromatogram
1.4 min
of
post- Fig. 4.9 (b) Graph of concentration of
acidification recovery of C13-HSL by strain C13-HSL shows the post-acidification
R3.7.
recovery of C4-HSL by strain R3.7.
Fig 4.6 (a), (b), 4.7 (a), (b), 4.8 (a), (b), 4.9 (a), (b) show the result of AHL inactivation
assay ( 0 h, 24h, 48h ) for selected AHLs at 4º C. After 48 hours, the samples were
acidified with HCl. The inactivated AHLs were recovered 24 hour post-acidification.
60
4.1.7 RRLC Analysis of Degradation of AHLs by Planococcus sp. strain L10.15
RRLC Analysis
of Degradation of AHLs by Planococcus sp. strain L10.15.
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 C A -2 0 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 C B -2 1 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 C C -2 2 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 C D -2 3 0 1 .D )
mAU
0h
200
175
150
125
100
75
50
24 h
25
48 h
72 h
0
0 .5
1
m in
1 .5
Fig. 4.10.1a RRLC analysis of synthetic C4-HSL (100µg) incubated with Planococcus
sp. strain L10.15, at 0-, 24-, 48-h and 72-h at 4°C.
DAD1
DAD1
DAD1
DAD1
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
2011-08-08
2011-08-08
2011-08-08
2011-08-08
11-51-42\2B A -0101.D )
11-51-42\2B A -0201.D )
11-51-42\2B A -0301.D )
11-51-42\2B A -0401.D )
mAU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
DAD1
DAD1
DAD1
DAD1
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
2011-08-08
2011-08-08
2011-08-08
2011-08-08
11-51-42\2B A -0501.D )
11-51-42\2B A -0601.D )
11-51-42\2B A -0701.D )
11-51-42\2B A -0801.D )
0
0.5
1
1.5
m in
0.5
1
m in
1.5
Fig.4.10.1b (left) and 4.10.1c (right) RRLC analysis of synthetic C4-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, 48-h and 72-h
at 4°C.
Legends:
Chromatogram of C4-HSL at 0-h
Chromatogram of C4-HSL at 24-h
Chromatogram of C4-HSL at 48-h
Chromatogram of C4-HSL at 72-h
61
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \C 2 W S _ C 7 C A 2 0 1 1 -0 8 -0 9 1 7 -0 6 -0 3 \1 A F -9 7 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \C 2 W S _ C 7 C A 2 0 1 1 -0 8 -0 9 1 7 -0 6 -0 3 \1 B F -9 8 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \C 2 W S _ C 7 C A 2 0 1 1 -0 8 -0 9 1 7 -0 6 -0 3 \1 C F -9 9 0 1 .D )
mAU
175
0h
150
125
24 h
100
75
50
25
48 h
0
0 .5
1
m in
1 .5
Figure 4.10.2a RRLC analysis of synthetic C5-HSL (100 µg) incubated with
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AA-0101.D )
A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 03-25-51\2AA-0501.D)
Planococcus
sp.(Cstrain
at 0-,) 24-, 48- h at DAD1
4°C.
D AD 1 A, Sig=210,4 R ef=360,100
2W S\C 2W S_C 7CL10.15,
A 2011-08-09 03-25-51\2AA-0201.D
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 03-25-51\2AA-0601.D)
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AA-0401.D )
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 03-25-51\2AA-0701.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2W S\C2W S_C7CA 2011-08-09 03-25-51\2AA-0801.D)
mAU
mAU
120
120
100
100
80
80
60
60
40
40
20
20
0
0
0.25
0.5
0.75
1
1.25
1.5
min
0.25
0.5
0.75
1
1.25
1.5
min
Fig.4 .10.2b (left) and 4.10.2c (right) RRLC analysis of synthetic C5-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C5-HSL at 0-h
Chromatogram of C5-HSL at 24-h
Chromatogram of C5-HSL at 48-h
62
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1AG-7601.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1BG-7701.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1CG-7801.D)
mAU
0h
200
175
150
24 h
125
100
75
50
48 h
25
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.10.3a RRLC analysis of synthetic C6-HSL (100µg) with Planococcus sp. strain
L10.15, at 0-, 24-, 48- h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
2011-08-09 03-25-51\2A B -1701.D )
2011-08-09 03-25-51\2A B -1801.D )
2011-08-09 03-25-51\2A B -1901.D )
2011-08-09 03-25-51\2A B -2001.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
m AU
2011-08-09 03-25-51\2AB-2101.D )
2011-08-09 03-25-51\2AB-2201.D )
2011-08-09 03-25-51\2AB-2301.D )
2011-08-09 03-25-51\2AB-2401.D )
m AU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
1
1.5
m in
Fig.4.10.3b (left) and 4.10.3c (right) RRLC analysis of synthetic C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C6-HSL at 0-h
Chromatogram of C6-HSL at 24-h
Chromatogram of C6-HSL at 48-h
63
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1AH-8001.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1BH-8101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 03-25-51\1DH-8301.D)
mAU
200
0h
175
24 h
150
125
48 h
100
75
50
25
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.10.4a RRLC analysis of synthetic 3-Oxo-C6-HSL (100µg) with Planococcus sp.
strain L10.15, at 0-, 24-, 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -4001.D )
2011-08-09 03-25-51\2AC -3301.D )
2011-08-09 03-25-51\2AC -3401.D )
2011-08-09 03-25-51\2AC -3501.D )
2011-08-09 03-25-51\2AC -3601.D )
m AU
mAU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.10.4b (left) and 4.10.4c (right) RRLC analysis of synthetic 3-Oxo-C6-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C6-HSL at 0-h
Chromatogram of 3-Oxo-C6-HSL at 24-h
Chromatogram of 3-Oxo-C6-HSL at 48-h
64
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \1 A I-8 4 0 1 .D )
0 3 -2 5 -5 1 \1 B I-8 5 0 1 .D )
0 3 -2 5 -5 1 \1 C I-8 6 0 1 .D )
0 3 -2 5 -5 1 \1 D I-8 7 0 1 .D )
mAU
100
0h
80
60
40
24 h
48 h
20
72 h
0
0 .5
1
m in
1 .5
Fig. 4.10.5a RRLC analysis of synthetic 3-Hydroxy-C6-HSL (100µg) with Planococcus
sp. strain L10.15, at 0-, 24-, 48- and 72-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5601.D )
2011-08-09 03-25-51\2A D -4901.D )
2011-08-09 03-25-51\2A D -5001.D )
2011-08-09 03-25-51\2A D -5101.D )
2011-08-09 03-25-51\2A D -5201.D )
m AU
mAU
80
80
60
60
40
40
20
20
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.10.5b (left) and 4.10.5c (right) RRLC analysis of synthetic 3-hydroxy-C6-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Hydroxy-C6-HSL at 0-h
Chromatogram of 3-Hydroxy-C6-HSL at 24-h
Chromatogram of 3-Hydroxy-C6-HSL at 48-h
Chromatogram of 3-Hydroxy-C6-HSL at 72-h
65
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1AA-8101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1CA-8301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1DA-8401.D)
mAU
0h
160
140
24 h
120
48 h
100
80
60
40
20
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.10.6a RRLC analysis of synthetic C7-HSL (100µg) with Planococcus sp. strain
L10.15, at 0-, 24-, 48-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0401.D )
mAU
175
m AU
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.10.6b (left) and 4.10.6c (right) RRLC analysis of synthetic C7-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C7-HSL at 0-h
Chromatogram of C7-HSL at 24-h
Chromatogram of C7-HSL at 48-h
66
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \1 A B -8 5 0 1 .D )
1 7 -0 6 -0 3 \1 B B -8 6 0 1 .D )
1 7 -0 6 -0 3 \1 C B -8 7 0 1 .D )
1 7 -0 6 -0 3 \1 D B -8 8 0 1 .D )
mAU
30
20
0h
10
24 h
48 h
0
72 h
-1 0
0 .5
1
m in
1 .5
Fig.4.10.7a RRLC analysis of synthetic C8-HSL (100µg) with Planococcus sp. strain
L10.15, at 0-, 24-, 48- and 72-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
2011-08-09 17-06-03\2A B -1701.D )
2011-08-09 17-06-03\2A B -1801.D )
2011-08-09 17-06-03\2A B -1901.D )
2011-08-09 17-06-03\2A B -2001.D )
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
mAU
m AU
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
m in
0.5
1
2011-08-09 17-06-03\2A B -2101.D )
2011-08-09 17-06-03\2A B -2201.D )
2011-08-09 17-06-03\2A B -2301.D )
2011-08-09 17-06-03\2A B -2401.D )
1.5
m in
Fig.4.10.7b (left) and 4.10.7c (right) RRLC analysis of synthetic C8-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C8-HSL at 0-h
Chromatogram of C8-HSL at 24-h
Chromatogram of C8-HSL at 48-h
Chromatogram of C8-HSL at 72-h
67
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \1 A C -8 9 0 1 .D )
1 7 -0 6 -0 3 \1 B C -9 0 0 1 .D )
1 7 -0 6 -0 3 \1 C C -9 1 0 1 .D )
1 7 -0 6 -0 3 \1 D C -9 2 0 1 .D )
mAU
0h
300
24 h
250
48 h
200
72 h
150
100
50
0
0 .5
1
m in
1 .5
Fig.4.10.8a RRLC analysis of synthetic 3-Oxo-C8-HSL (100µg) with Planococcus sp.
strain L10.15, at 0-, 24-, 48- and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -4001.D )
2011-08-09 17-06-03\2AC -3301.D )
2011-08-09 17-06-03\2AC -3401.D )
2011-08-09 17-06-03\2AC -3501.D )
2011-08-09 17-06-03\2AC -3601.D )
m AU
mAU
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.10.8b (left) and 4.10.8c (right) RRLC analysis of synthetic 3-Oxo-C8-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C8-HSL at 0-h
Chromatogram of 3-Oxo-C8-HSL at 24-h
Chromatogram of 3-Oxo-C8-HSL at 48-h
Chromatogram of 3-Oxo-C8-HSL at 72-h
68
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1AD-9301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1BD-9401.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-09 17-06-03\1CD-9501.D)
mAU
70
60
50
0h
40
30
24 h
20
48 h
10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.10.9a RRLC analysis of synthetic 3-Hydroxy-C8-HSL (100µg) with Planococcus
sp. strain L10.15, at 0-, 24-, 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
2011-08-09 17-06-03\2AD -5301.D )
2011-08-09 17-06-03\2AD -5401.D )
2011-08-09 17-06-03\2AD -5501.D )
2011-08-09 17-06-03\2AD -5601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
m AU
m AU
40
40
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
m in
0.5
2011-08-09 17-06-03\2AD -5301.D )
2011-08-09 17-06-03\2AD -5401.D )
2011-08-09 17-06-03\2AD -5501.D )
2011-08-09 17-06-03\2AD -5601.D )
1
1.5
m in
Fig.4.10.9b (left) and 4.10.9c (right) RRLC analysis of synthetic 3- Hydroxy -C8-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Hydroxy-C8-HSL at 0-h
Chromatogram of 3-Hydroxy-C8-HSL at 24-h
Chromatogram of 3-Hydroxy-C8-HSL at 48-h
69
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
0 3 -2 6 -0 1 \2 A G -2 1 0 1 .D )
0 3 -2 6 -0 1 \2 B G -2 2 0 1 .D )
0 3 -2 6 -0 1 \2 C G -2 3 0 1 .D )
0 3 -2 6 -0 1 \2 D G -2 4 0 1 .D )
mAU
0h
70
24 h
60
48 h
50
72 h
40
30
20
10
0
0 .5
1
1 .5
2 .5 m in
2
Fig.4.10.10a RRLC analysis of synthetic C9-HSL (100µg) with Planococcus sp. strain
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AA-0101.D )
L10.15,
at 0-, 24-, 48- and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AA-0201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AA-0301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AA-0401.D )
m AU
m AU
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2011-08-11 03-26-01\2AA-0501.D )
2011-08-11 03-26-01\2AA-0601.D )
2011-08-11 03-26-01\2AA-0701.D )
2011-08-11 03-26-01\2AA-0801.D )
0
0.5
1
1.5
2
2.5
m in
0.5
1
1.5
2
2.5
m in
Fig.4.10.10b (left) and 4.10.10c (right) RRLC analysis of synthetic C9-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-,48-h and -72h
at 4°C.
Legends:
Chromatogram of C9-HSL at 0-h
Chromatogram of C9-HSL at 24-h
Chromatogram of C9-HSL at 48-h
Chromatogram of C9-HSL at 72-h
70
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
R ef = 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \T E S T
S \T E S T
S \T E S T
S \T E S T
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
2 0 1 1 -0 8 -1 1
0 3 -2 6 -0 1 \2 A H -4 5 0 1 .D )
0 3 -2 6 -0 1 \2 B H -4 6 0 1 .D )
0 3 -2 6 -0 1 \2 C H -4 7 0 1 .D )
0 3 -2 6 -0 1 \2 D H -4 8 0 1 .D )
mAU
70
0h
60
24 h
50
48 h
40
72 h
30
20
10
0
1
2
m in
3
Fig.4.10.11a RRLC analysis incubated of synthetic C10-HSL (100µg) with
Planococcus sp. strain L10.15, at 0-, 24-, 48- and 72- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 03-26-01\2AB-2501.D )
2011-08-11 03-26-01\2AB-2601.D )
2011-08-11 03-26-01\2AB-2701.D )
2011-08-11 03-26-01\2AB-2801.D )
m AU
mAU
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
2011-08-11 03-26-01\2AB-2901.D )
2011-08-11 03-26-01\2AB-3001.D )
2011-08-11 03-26-01\2AB-3101.D )
2011-08-11 03-26-01\2AB-3201.D )
0
0
1
2
3
m in
1
2
3
min
Fig. 4.10.11b (left) and 4.10.11c (right) RRLC analysis of synthetic C10-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-,48-h and -72h
at 4°C.
Legends:
Chromatogram of C10-HSL at 0-h
Chromatogram of C10-HSL at 24-h
Chromatogram of C10-HSL at 48-h
Chromatogram of C10-HSL at 72-h
71
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 11-11-10\2BI-7001.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 11-11-10\2CI-7101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\TEST 2011-08-11 11-11-10\2DI-7201.D)
mAU
200
175
150
0h
125
100
24 h
75
48 h
50
25
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Fig.4.10.12a RRLC analysis of synthetic 3-Oxo-C12-HSL (100µg) with Planococcus
sp. strain L10.15, at 0-, 24-, 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 11-11-10\2AC -4901.D )
2011-08-11 11-11-10\2AC -5001.D )
2011-08-11 11-11-10\2AC -5101.D )
2011-08-11 11-11-10\2AC -5201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AC -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AC -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AC -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 11-11-10\2AC -5601.D )
m AU
200
mAU
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.10.12b (left) and 4.10.12c (right) RRLC analysis of synthetic 3-oxo-C12-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C12-HSL at 0-h
Chromatogram of 3-Oxo-C12-HSL at 24-h
Chromatogram of 3-Oxo-C12-HSL at 48-h
72
4.1.8 Quantitative Analysis of AHL Degradation by Planococcus sp. strain L10.15
Table 4.2 Estimation of activity by Planococcus sp. strain L10.15 of various AHLs
Types of AHL
E.A*
C4-HSL
55.88
C5-HSL
31.25
C6-HSL
27.22
3-oxo-C6-HSL
6.38
3-hydroxy-C6-HSL
21.33
C7-HSL
8.48
C8-HSL
8.87
3-oxo-C8-HSL
8.32
3-hydroxy-C8-HSL
10.25
C10-HSL
7.68
3-Oxo-C12-HSL
5.31
*EA, estimated activity, expressed as ng AHL degraded h-1(105CFU/ml)-1 at 4°C
73
Fig. 4.11 shows the representative graphs to show the degradation of C4-HSL by
Planococcus sp. strain L10.15.
Fig. 4.12 shows the representative graphs to show the degradation of 3-oxo-C6-HSL by
Planococcus sp. strain L10.15
74
Fig.4.13 shows the representative graphs to show the degradation of 3-hydroxy-C6HSL by Planococcus sp. strain L10.15.
Fig. 4.14 shows the representative graphs to show the degradation of C8-HSL by
Planococcus sp. strain L10.15.
75
Figures 4.11, 4.12, 4.13 and 4.14 are the representative graphs to show the
degradation of AHLs from different group ( short chain AHL, long chain AHL, with or
without substitution group) by Planococcus sp. strain L10.15. These graphs show the
amount of AHL for AHL inactivation assay from 0-72 h. PBS buffer (100mM, pH 6.5)
and E. coli DH5α were used as negative controls for the assay.
From the quantitative data (Table 4.2), Planococcus sp. strain L10.15 was most
effectively to degrade C4-HSL and C5-HSL. Strain L10.15 can even degrade 3-heptyl4-quinolone (HHQ) .
.
76
4.1.9 Determination of AHL Degradation via Lactonase Activity by Planococcus sp.
strain L10.15
Fig. 4.15 (a) Chromatogram of post-
Fig. 4.15 (b) Graph of concentration
acidification recovery of C6-HSL by
of C6-HSL shows the post-
Planococcus sp. strain L10.15
acidification recovery of C4-HSL by
Planococcus sp. strain L10.15
Fig 4.16 (a) Chromatogram of post- Fig 4.16 (b) Graph of concentration of
acidification recovery of C7-HSL by C7-HSL
Planococcus sp. strain L10.15
shows
the
post-acidification
recovery of C4-HSL by Planococcus sp.
77
Fig. 4.17 (a) Chromatogram of post-
Fig.4.17 (b) Graph of concentration of
acidification recovery of 3-hydroxy-C8-
3- hydroxy-C8-HSL shows the post-
HSL by Planococcus sp. strain L10.15
acidification recovery of C4-HSL by
Planococcus sp. strain L10.15
Fig.4.15 (a), (b), 4.16 (a), (b), 4.17 (a), (b), show the result of AHL inactivation
assay ( 0 h, 24h, 48h ) for selected AHLs at 4º C. After 48 hours, the samples were
acidified with HCl. The inactivated AHLs were recovered 24 hour post-acidification.
78
4.1.10 Degradation of various AHLs by Flavobacterium sp. strain Ln5b.12
RRLC Analysis of Degradation of AHLs by Flavobacterium sp. strain Ln5b.12
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 B A -1 7 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 B C -1 8 0 1 .D )
D A D 1 A , S ig = 2 1 0 ,4 R ef = 3 6 0 ,1 0 0 (C 2 W S \T E S T 2 0 1 1 -0 8 -0 8 1 1 -5 1 -4 2 \2 B D -1 9 0 1 .D )
mAU
200
150
0h
100
24 h
50
48 h
0
0 .2 5
0 .5
0 .7 5
1
1 .2 5
1 .5
m in
1 .7 5
Fig.4.18.1a RRLC analysis of synthetic C4-HSL (100µg) with Flavobacterium sp.
strain Ln5b.12, E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-08 11-51-42\2BA-0101.D )
2011-08-08 11-51-42\2BA-0201.D )
2011-08-08 11-51-42\2BA-0301.D )
2011-08-08 11-51-42\2BA-0401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
m AU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
2011-08-08 11-51-42\2BA-0501.D )
2011-08-08 11-51-42\2BA-0601.D )
2011-08-08 11-51-42\2BA-0701.D )
2011-08-08 11-51-42\2BA-0801.D )
1
1.5
min
Fig. 4.18.1b (left) and 4.18.1c (right) RRLC analysis of synthetic C4-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C4-HSL at 0-h
Chromatogram of C4-HSL at 24-h
Chromatogram of C4-HSL at 48-h
79
DAD1 A, Sig=210,4 Ref=360,100 (C2WS_C7CA 2011-01-19 05-25-02\1CH-2601.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS_C7CA 2011-01-19 05-25-02\1CI-2701.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS_C7CA 2011-01-19 05-25-02\1DA-2801.D)
mAU
0h
350
300
250
24 h
200
150
48 h
100
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.18.2a RRLC analysis of synthetic C6-HSL (100µg) with Flavabacterium sp.
1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1701.D )
strainDD ADADLn5b.12,
at 0-, 24-, 48- h at 4°C.
1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2201.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-1901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AB-2001.D )
m AU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig. 4.18.2b (left) and 4.18.2c (right) RRLC analysis of synthetic C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C6-HSL at 0-h
Chromatogram of C6-HSL at 24-h
Chromatogram of C6-HSL at 48-h
80
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \1 B B -6 9 0 1 .D )
0 3 -2 5 -5 1 \1 A B -6 8 0 1 .D )
0 3 -2 5 -5 1 \1 C B -7 0 0 1 .D )
0 3 -2 5 -5 1 \1 D B -7 1 0 1 .D )
mAU
0h
350
300
24 h
250
48 h
200
72 h
150
100
50
0
0 .2 5
0 .5
0 .7 5
1
1 .2 5
1 .5
1 .7 5
m in
Fig. 4.18.3a RRLC analysis of synthetic 3- Oxo-C6-HSL (100µg) with Flavabacterium
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3701.D )
sp. strain
Ln5b.12,
24-,
4872 h at 4°C.
D AD 1 A, Sig=210,4
R ef=360,100 (C 2W S\C 2Wat
S_C 7C0-,
A 2011-08-09
03-25-51\2AC
-3401.Dand
)
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -4001.D )
mAU
mAU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
min
0.5
1
1.5
min
Fig. 4.18.3b (left) and 4.18.3c (right) RRLC analysis of synthetic 3- Oxo-C6-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3- Oxo-C6-HSL at 0-h
Chromatogram of 3- Oxo-C6-HSL at 24-h
Chromatogram of 3- Oxo-C6-HSL at 48-h
Chromatogram of 3- Oxo-C6-HSL at 72-h
81
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
0 3 -2 5 -5 1 \1 A C -7 2 0 1 .D )
0 3 -2 5 -5 1 \1 B C -7 3 0 1 .D )
0 3 -2 5 -5 1 \1 C C -7 4 0 1 .D )
0 3 -2 5 -5 1 \1 D C -7 5 0 1 .D )
mAU
0h
80
60
24 h
40
48 h
72 h
20
0
0 .2 5
0 .5
0 .7 5
1
1 .2 5
1 .5
1 .7 5
m in
Fig.4.18.4a RRLC analysis of synthetic 3-Hydroxy-C6-HSL with Flavabacterium sp.
strain Ln5b.12, at 0-, 24-, 48- and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AD -5601.D )
2011-08-09 03-25-51\2AD -4901.D )
2011-08-09 03-25-51\2AD -5001.D )
2011-08-09 03-25-51\2AD -5101.D )
2011-08-09 03-25-51\2AD -5201.D )
m AU
mAU
80
80
60
60
40
40
20
20
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.18.4b (left) and 4.18.4c (right) RRLC analysis of synthetic 3-hydroxy-C6-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, 48-h
and 72-h at 4°C.
Legends:
Chromotogram of 3-Hydroxy-C6-HSL at 0-h
Chromotogram of 3-Hydroxy-C6-HSL at 24-h
Chromotogram of 3-Hydroxy-C6-HSL at 48-h
Chromotogram of 3-Hydroxy-C6-HSL at 72-h
82
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \2 B F -6 6 0 1 .D )
1 7 -0 6 -0 3 \2 A F -6 5 0 1 .D )
1 7 -0 6 -0 3 \2 C F -6 7 0 1 .D )
1 7 -0 6 -0 3 \2 D F -6 8 0 1 .D )
mAU
0h
175
150
125
24 h
100
75
48 h
50
72 h
25
0
0 .2 5
0 .5
0 .7 5
1
1 .2 5
1 .5
1 .7 5
m in
Fig.4.18.5a RRLC analysis of synthetic C7-HSL (100µg) with Flavabacterium sp.
strain Ln5b.12, at 0-, 24-, 48- and 72-h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0601.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AA-0801.D )
2011-08-09 17-06-03\2AA-0101.D )
2011-08-09 17-06-03\2AA-0201.D )
2011-08-09 17-06-03\2AA-0301.D )
2011-08-09 17-06-03\2AA-0401.D )
m AU
175
mAU
175
150
150
125
125
100
100
75
75
50
50
25
25
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.18.5b (left) and 4.18.5c (right) RRLC analysis of synthetic C7-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, 48-h and 72-h
at 4°C.
Legends:
Chromatogram of C7-HSL at 0-h
Chromatogram of C7-HSL at 24-h
Chromatogram of C7-HSL at 48-h
Chromatogram of C7-HSL at 72-h
83
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
2 0 1 1 -0 8 -0 9
1 7 -0 6 -0 3 \2 B G -7 0 0 1 .D )
1 7 -0 6 -0 3 \2 A G -6 9 0 1 .D )
1 7 -0 6 -0 3 \2 C G -7 1 0 1 .D )
1 7 -0 6 -0 3 \2 D G -7 2 0 1 .D )
mAU
0h
20
24 h
15
10
48 h
5
72 h
0
-5
-1 0
-1 5
0 .2
0 .4
0 .6
0 .8
1
1 .2
1 .4
m in
Fig.4.18.6a RRLC analysis of synthetic C8-HSL (100 µg) with Flavabacterium sp.
strain Ln5b.12, at 0-, 24-, 48-and 72-h at 4°C.
DAD1
DAD1
DAD1
DAD1
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
2011-08-09
2011-08-09
2011-08-09
2011-08-09
17-06-03\2A B -1701.D )
17-06-03\2A B -1801.D )
17-06-03\2A B -1901.D )
17-06-03\2A B -2001.D )
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
mAU
m AU
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
m in
0.5
1
2011-08-09 17-06-03\2A B -2101.D )
2011-08-09 17-06-03\2A B -2201.D )
2011-08-09 17-06-03\2A B -2301.D )
2011-08-09 17-06-03\2A B -2401.D )
1.5
m in
Fig.4.18.6b (left) and 4.18.6c (right) RRLC analysis of synthetic C8-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, 48-h and 72-h
at 4°C.
Legends:
Chromatogram of C8-HSL at 0-h
Chromatogram of C8-HSL at 24-h
Chromatogram of C8-HSL at 48-h
Chromatogram of C8-HSL at 72-h
84
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-10 08-20-35\2AH-0101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-10 08-20-35\2BH-0201.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-08-10 08-20-35\2CH-0301.D)
mAU
350
0h
300
250
200
24 h
150
48 h
100
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
min
1.8
Fig.4.18.7a RRLC analysis of synthetic 3-Oxo-C8-HSL (100µg) with Flavabacterium
sp. strain Ln5b.12, at 0-, 24-, and 48-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AC -4001.D )
2011-08-09 17-06-03\2A C -3301.D )
2011-08-09 17-06-03\2A C -3401.D )
2011-08-09 17-06-03\2A C -3501.D )
2011-08-09 17-06-03\2A C -3601.D )
m AU
mAU
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.18.7b (left) and 4.18.7c (right) RRLC analysis of synthetic 3-Oxo-C8-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Oxo-C8-HSL at 0-h
Chromatogram of 3-Oxo-C8-HSL at 24-h
Chromatogram of 3-Oxo-C8-HSL at 48-h
85
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
S ig = 2 1 0 ,4
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
R ef= 3 6 0 ,1 0 0
(C 2 W
(C 2 W
(C 2 W
(C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S \C 2 W
S _C 7C A
S _C 7C A
S _C 7C A
S _C 7C A
2 0 1 1 -0 8 -1 0
2 0 1 1 -0 8 -1 0
2 0 1 1 -0 8 -1 0
2 0 1 1 -0 8 -1 0
0 8 -2 0 -3 5 \2 B I-0 6 0 1 .D )
0 8 -2 0 -3 5 \2 A I-0 5 0 1 .D )
0 8 -2 0 -3 5 \2 C I-0 7 0 1 .D )
0 8 -2 0 -3 5 \2 D I-0 8 0 1 .D )
mAU
40
0h
30
24 h
20
48 h
10
72 h
0
-1 0
0 .2 5
0 .5
0 .7 5
1
1 .2 5
1 .5
m in
1 .7 5
Fig.4.18.8a RRLC analysis of synthetic 3-Hydroxy-C8-HSL with Flavabacterium sp.
strain Ln5b.12, at 0-, 24-, 48- and 72- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A
2011-08-09 17-06-03\2AD -4901.D )
2011-08-09 17-06-03\2AD -5001.D )
2011-08-09 17-06-03\2AD -5101.D )
2011-08-09 17-06-03\2AD -5201.D )
m AU
mAU
40
40
30
30
20
20
10
10
0
0
-10
-10
0.5
1
1.5
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5301.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5401.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5501.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 17-06-03\2AD -5601.D )
m in
0.5
1
1.5
min
Fig.4.18.8b (left) and 4.18.8c (right) RRLC analysis of synthetic 3-hydroxy-C8-HSL
(100µg) incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and
48-h at 4°C.
Legends:
Chromatogram of 3-Hydroxy-C8-HSL of 0-h
Chromatogram of 3-Hydroxy-C8-HSL of 24-h
Chromatogram of 3-Hydroxy-C8-HSL of 48-h
Chromatogram of 3-Hydroxy-C8-HSL of 72-h
86
DAD1
DAD1
DAD1
DAD1
A,
A,
A,
A,
S ig= 210,4
S ig= 210,4
S ig= 210,4
S ig= 210,4
R ef= 360,100
R ef= 360,100
R ef= 360,100
R ef= 360,100
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
(C 2W S \T E S T
2011-08-11
2011-08-11
2011-08-11
2011-08-11
03-26-01\2A E -4101.D )
03-26-01\2B E -4201.D )
03-26-01\2C E -4301.D )
03-26-01\2D E -4401.D )
mAU
0h
70
60
50
40
24 h
30
48 h
20
72 h
10
0
0.5
1
1.5
m in
2
Fig.4.18.9a RRLC analysis of synthetic C10-HSL (100 µg) with Flavabacterium sp.
strain Ln5b.12, at 0-, 24-, 48- and 72-h at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \T E S T
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AB-2901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AB-3001.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AB-3101.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST 2011-08-11 03-26-01\2AB-3201.D )
2011-08-11 03-26-01\2A B -2501.D )
2011-08-11 03-26-01\2A B -2601.D )
2011-08-11 03-26-01\2A B -2701.D )
2011-08-11 03-26-01\2A B -2801.D )
m AU
mAU
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
1
2
3
m in
1
2
3
min
Fig.4.18.9b (left) and 4.18.9c (right) RRLC analysis of synthetic C10-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-,48- and 72-h at
4°C.
Legends:
Chromatogram of C10-HSL at 0-h
Chromatogram of C10-HSL at 24-h
Chromatogram of C10-HSL at 48-h
Chromatogram of C10-HSL at 72-h
87
4.1.11 Quantitative Analysis of AHL Degradation by Flavabacterium sp. strain
Ln5b.12
Table 4.3 Estimation of activity by Flavabacterium sp. strain Ln5b.12 of various
AHLs
Types of AHL
E.A*
C4-HSL
21.24
C5-HSL
3.65
C6-HSL
3.26
3-oxo-C6-HSL
6.85
3-hydroxy-C6-HSL
13.27
C7-HSL
20.97
C8-HSL
20.43
3-oxo-C8-HSL
14.95
3-hydroxy-C8-HSL
22.10
C9-HSL
11.48
C10-HSL
15.44
*EA, estimated activity, expressed as ng AHL degraded h-1(105CFU/ml)-1 at 4°C
88
Fig.4.19 shows the representative graphs to show the degradation of C4-HSL by
Flavabacterium sp. strain Ln5b.12
Fig .4.20 shows the representative graphs to show the degradation of 3-hydroxy-C6HSL by Flavabacterium sp. strain Ln5b.12
89
Fig. 4.21 shows the representative graphs to show the degradation of C8-HSL by
Flavabacterium sp. strain Ln5b.12
Fig. 4.22 shows the representative graphs to show the degradation of 3-oxo-C8-HSL by
Flavobacterium sp. strain Ln5b.12
Figures 3.19, 3.20, 3.21 and 3.22 are the representative graphs to show the
degradation of AHLs from different group ( short chain AHL, medium chain AHL, with
or without substitution group) by Flavobacterium sp. strain Ln5c.12b. These graphs
90
show the amount of AHL for AHL inactivation assay from 0-72 h. PBS buffer (100mM,
pH 6.5) and E.coli were used as negative control for the assay.
4.2 Enrichment and isolation of soil bacteria from various locations from
Antarctica
Enrichment of QQ bacteria was carried out using modified KG medium
containing synthetic AHLs, 3-oxo-C6-HSL, C7-HSL or C12-HSL as described in
Section 2.2. Modified KG medium supplied with C12-HSL as sole source of energy
and nitrogen, which was inoculated with soil sample of three ecologically different sites
from Casey Station (Table 2.1), became turbid after 3 weeks. An amount of 21 bacteria
with different morphology were then isolated. For soil samples collected near Rothera
Research Station (Table 2.2), (modified KG medium was supplied with either 3-oxoC6-HSL or C7-HSL as carbon and nitrogen sources), the medium become turbid
(compared to the negative control), and 46 bacteria were isolated.
4.2.1 Soil physical properties and isolation of QQ bacteria
The pH, salinity and moisture of the soil were analyzed (Table 4.4).
Table 4.4 showed the environment properties of soil sample collected from
different sites of Casey Research Station
N/A
Moisture
(%)
8.99
Thala Valley
N/A
ASPA 136
N/A
Location
Elevation
(m)
Browning
Peninsular
pH
6.22
Salinity
(µS)
24.62
Temperature
(°C)
N/A
6.13
7.56
657.5
N/A
24.71
4.78
189.70
N/A
91
Table 4.5 shows the environment properties of the soil samples collected from
Rothera Research Station and the isle near-by
7.4
Moisture
(%)
6.239
5.21
Salinity
(µS)
49.4
Temperature
(°C)
8.7
Lagoon Island-4
10.3
1.206
7.48
192.9
8.5
Leonie Island-4
1.8
16.918
6.34
86.4
4
Leonie Island-5
11.1
13.082
7.41
31.3
4
Rothera Point-3
3
29.167
6.74
100.9
8.2
Anchorage Island-4
17.3
84.402
4.61
84.8
13.5
Anchorage Island5
27.2
6.034
3.51
80.2
7.0
Anchorage Island6
16.6
14.259
2.4
460
6.5
Donnelly Island-1
23.7
4.189
7.92
156.8
5.5
Killingbeck Island1
4
11.527
7.97
168.5
4.7
Location
Elevation
(m)
Lagoon Island-3
pH
92
4.2.2 16S rDNA gene identification and phylogenetic analysis of Pseudomonas spp.
isolated from Thala Valley
Four out of five bacteria strains that were found in Thala Valley are
Pseudomonas spp. The 16S rDNA gene-based phylogenetic tree (Fig. 4.23) was rooted
with Pseudomonas aeruginosa strain MW3A (GenBank accession number GQ 180117)
as outgroup. Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of
the 16S rDNA gene supported strain T7A (gene accession number: KC479345) is a
strain of Pseudomonas putida, strain T7C (gene accession number: KC433634) is
Pseudomonas antarctica, strain T7D (gene accession number: KC433635) is a strain of
Pseudomonas frederiksbergensis. The nearest identity found for strain T7B (gene
accession number: KC433633) is Pseudomonas fluorescens.
100 T7C
Pseudomonas antarctica isolate FB27 (AM933518)
87
T7B
92
88
Pseudomonas fluorescens strain P69 (AY973265)
T7A
100 Pseudomonas putida gene for 16S rRNA strain: NBRC 12653 (AB680304)
T7D
100 Pseudomonas frederiksbergensis strain B62 (EU169158)
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.23 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Thala Valley with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
93
4.2.3 16S rDNA gene identification and phylogenetic analysis of Pseudomonas spp.
isolated from ASPA 136
Five bacteria strains that were found in ASPA 136 are Pseudomonas spp. The
16S rDNA gene-based phylogenetic tree (Fig. 4.24) was rooted with Pseudomonas
aeruginosa strain MW3A (GenBank accession number GQ 180117) as outgroup.
Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S
rDNA gene supported strain the nearest identity for strain B122 (gene accession number:
KC433656) is Pseudomonas migulae, the nearest identity for strain 6PC5 (gene
accession number: KC433603) is Pseudomonas antarctica, the nearest identity for
strain 6PC2 (gene accession number: KC433599) is Pseudomonas psychrophila, the
nearest identity for strain B62 (gene accession number: KC433608) is Pseudomonas
syringae and the nearest identity found for strain 621(gene accession number:
KC433600) is Pseudomonas fluorescens.
96 621
Pseudomonas fluorescens strain P69 (AY973265)
45
B122
50
95 Pseudomonas migulae strain D67 (JN228320)
Pseudomonas brenneri isolate FB30 (AM933521)
6PC5
41
99
96
Pseudomonas antarctica Asd M6-3 (FM955872)
6PC2
Pseudomonas psychrophila strain E-3 (NR 028619)
91
B62
90 Pseudomonas syringae strain 1 (AM184090)
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.24 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from ASPA 126 with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
94
4.2.4 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Browning Peninsular
Six of the bacteria strains found in Browning Peninsular were Pseudomonas spp.
The 16S rDNA gene-based phylogenetic tree (Fig. 4.25) was rooted with Pseudomonas
aeruginosa strain MW3A (GenBank accession number GQ 180117) as outgroup.
Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S
rDNA gene supported strain the nearest identity for strain B7C (gene accession number:
KC433606) and B8D (gene accession number: KC433607) is Pseudomonas migulae,
the nearest identity for strain B7E (gene accession number: KC433647) is Pseudomonas
gerssardii, the nearest identity for strain B7B (gene accession number: KC433652) is
Pseudomonas frederiksbergensis, the nearest identity for strain B8A (gene accession
number: KC433602) is Pseudomonas lini and the nearest identity found for strain B7A
(gene accession number: KC433605) is Pseudomonas brenneri.
80
B7C
Pseudomonas migulae strain D67 (JN228320)
38
B7E
97
41
85
Pseudomonas gessardii strain CIP 105469 (NR 024928)
B7B
Pseudomonas frederiksbergensis strain DSM 13022T (FR750403)
47
B8D
44
86
Pseudomonas migulae strain CT14 (EU111725)
B8A
97
Pseudomonas lini isolate PD 15 (DQ377756)
B7A
92
Pseudomonas brenneri isolate FB30 (AM933521)
Pseudomonas psychrophila strain E-3 (NR 028619)
0.005
Fig. 4.25 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Browning Peninsular with closely related taxa. Bar represents
evolutionary distance as 0.01 change per nucleotide position. Bootstrap values (%) over
50% from 1,000 replications are shown.
95
4.2.5 16S rDNA gene identification and phylogenetic analysis of Arthrobacter sp.
isolated from Casey Station
Strain T7E was the only of Arthrobacter sp. found in Thala Valley. The 16S
rDNA gene-based phylogenetic tree (Fig. 4.26) was rooted with Arthrobacter oxydans
strain 92-0600 (GenBank accession number EU977757) as outgroup. Phylogenetic
analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S rDNA gene
showed that the nearest identity for strain T7E (gene accession number: KC433651) is
Arthrobacter psychrolactophilus strain KNOUC403.
71
T7E
63 Arthrobacter psychrolactophilus strain KNOUC403 16S ribosomal RNA gene partial sequence (HQ610444)
Arthrobacter psychrochitiniphilus gene for 16S rRNA strain: JCM 13874(AB588633)
Arthrobacter stackebrandtii strain 2P03PA 16S ribosomal RNA gene (EU977757)
Arthrobacter oxydans strain 92-0600 16S ribosomal RNA gene (EU086811)
0.001
Fig. 4.26 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Arhtrobacter strains that
were isolated from Thala Valley with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
96
4.2.6 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Lagoon Island
Five out of six bacteria strains that were found in Lagoon Island are
Pseudomonas spp. The 16S rDNA gene-based phylogenetic tree (Fig. 4.27) was rooted
with Pseudomonas aeruginosa strain MW3A (GenBank accession number GQ 180117)
as outgroup. Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of
the 16S rDNA gene supported that the nearest identity for strain L10.4 (gene accession
number: KC505201) and L10.10 (gene accession number: KC433618) are strain of
Pseudomonas fragi, strain L1R3.6 (gene accession number: KC433616) is
Pseudomonas migulae, the nearest identity for strain L1R3.5 (gene accession number:
KC433653) is strain of Pseudomonas frederiksbergensis. The nearest identity found for
strain L10.1 (gene accession number: KC433614) is Pseudomonas fluorescens.
Fig. 4.27 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Lagoon Island with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
97
4.2.7 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Leonie Island
Five out of the nine bacteria strains that were found in Leonie Island are
Pseudomonas spp. The 16S rDNA gene-based phylogenetic tree (Fig. 4.28) was rooted
with Pseudomonas aeruginosa strain MW3A (GenBank accession number GQ 180117)
as outgroup. Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of
the 16S rDNA gene supported that the nearest identity for strain Ln5C.7 (gene
accession number: KC433645) strain is Pseudomonas fragi,the nearest identity fo strain
Ln5C.8 (gene accession number: KC433643) is Pseudomonas brenneri, the nearest
identity for strain Ln4B.8 (gene accession number: KC433642) is strain of
Pseudomonas frederiksbergensis, the nearest identity found for strain Ln4B.7 (gene
accession number: KC433641) is Pseudomonas fluorescens and the nearest identity
found for strain Ln4B.12g (gene accession number: KC433644) is Pseudomonas
antarctica.
86
92
Ln5C.8
Pseudomonas brenneri isolate FB30 (AM933521)
Pseudomonas migulae strain CT14 (EU111725)
34
Ln4B.7
74
Pseudomonas fluorescens strain P69 (AY973265)
88
73 Pseudomonas extremaustralis strain CT14-3 (AJ583501)
Pseudomonas veronii strain:INA06 (AB056120)
99
41
Pseudomonas marginalis strain NZCX27 (AF364098)
Ln4B.12g
66
91
Pseudomonas antarctica Asd M6-3 (FM955872)
Pseudomonas mandelii strain CIP 105273 (NR 024902)
Pseudomonas frederiksbergensis strain DSM 13022T
99
72
93
Ln4B.8
Pseudomonas frederiksbergensis strain B62 (EU169158)
Ln5c.7
100
Pseudomonas fragi gene for 16S ribosomal RNA strain: JCM 5418 (AB685630)
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.28 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Leonie Island with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
98
4.2.8 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Rothera Research Station
Four out of five bacteria strains that were found in Rothera Research Station
(Rothera Point) are Pseudomonas spp. except for strain R3.7. The 16S rDNA genebased phylogenetic tree (Fig. 4.29) was rooted with Pseudomonas aeruginosa strain
MW3A (GenBank accession number GQ 180117) as outgroup. Phylogenetic analysis
using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S rDNA gene supported that
the nearest identity for strain R3.12 (gene accession number: KC433650) is strain
Pseudomonas antarctica, strain R3.8 (gene accession number: KC433630) is
Pseudomonas syringae, and the nearest identity found for strain R3.9 (gene accession
number: KC433632) and R3.1B (gene accession number: KC433649) are Pseudomonas
fluorescens.
90 R3.12
Pseudomonas antarctica isolate FB27 (AM933518)
87
R3.9
51
63 Pseudomonas fluorescens strain MS300 16S ribosomal RNA gene (HQ589333)
78
Pseudomonas migulae strain R-20803 (AM114525)
Pseudomonas migulae strain D67 (JN228320)
R3.1B
86
97 Pseudomonas fluorescens strain LMG 14674 (GU198124)
Pseudomonas taetrolens gene for 16S rRNA strain: NBRC 3460 (AB680089)
R3.8
95
96
Pseudomonas syringae partial 16S rRNA gene isolate Lz4W (AJ576247)
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.29 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Rothera Research Station with closely related taxa. Bar represents
evolutionary distance as 0.01 change per nucleotide position. Bootstrap values (%) over
50% from 1,000 replications are shown.
99
4.2.9 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Donelly Island
Among all the bacteria found in Donelly Island, only one strain namely strain
D1.1 possesses QS activities. Not strain with QQ activity was found in Donelly Island.
The 16S rDNA gene-based phylogenetic tree (Fig. 4.30) was rooted with Moraxella
cuniculi strain CCUG 2154 (GenBank accession number NR 041695) as outgroup.
Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S
rDNA gene supported that the nearest identity for strain D1.1 (gene accession number:
KC433610) is strain Psychrobacter cryohalelentis.
98 D1.1
79
69
Psychrobacter cryohalolentis strain KOPRI 22219 16S ribosomal RNA gene (EU090718)
Psychrobacter fozii 16S ribosomal RNA gene (AY771717)
Psychrobacter arcticus 273-4 strain 273-4 16S ribosomal RNA (NR 042907)
Psychrobacter luti strain NF11 16S ribosomal RNA (NR 025532)
Moraxella cuniculi strain CCUG 2154 16S ribosomal RNA (NR 041695)
0.005
Fig. 4.30 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Donelly Island with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
100
4.2.10 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Killinbeck Island
Three bacteria strains found in Killingbeck Island were identified as
Pseudomonas spp. The 16S rDNA gene-based phylogenetic tree (Fig. 4.31) was rooted
with Pseudomonas aeruginosa strain MW3A (GenBank accession number GQ 180117)
as outgroup. Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of
the 16S rDNA gene supported strain the nearest identity for strain K3R3.2 (gene
accession number: KC433622) is Pseudomonas migulae, the nearest identity for strain
K3.4 (gene accession number: KC433648) is Pseudomonas mandelii, and the nearest
identity found for strain K3R3.1A (gene accession number: KC433646) is
Pseudomonas syringae.
98 K3R3.1A
100
Pseudomonas syringae partial 16S rRNA gene isolate Lz4W (AJ576247)
Pseudomonas taetrolens gene for 16S rRNA strain: NBRC 3460 (AB680089)
43
K3.4
100 Pseudomonas mandelii strain CIP 105273 (NR 024902)
Pseudomonas collierea partial 16S rRNA gene strain PR212T (AM421016)
98
50
Pseudomonas gessardii gene for 16S rRNA strain: NBRC 101045 (AB681347)
K3R3.2
96 Pseudomonas migulae strain D67 16S ribosomal RNA gene (JN2283200
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.31 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Killingbeck Island with closely related taxa. Bar represents
evolutionary distance as 0.01 change per nucleotide position. Bootstrap values (%) over
50% from 1,000 replications are shown.
101
4.2.11 16S rDNA gene identification and phylogenetic analysis of Pseudomonas sp.
isolated from Anchorage Island
Five bacteria strains found in Anchorage Island were Pseudomonas spp. The
16S rDNA gene-based phylogenetic tree (Fig. 4.32) was rooted with Pseudomonas
aeruginosa strain MW3A (GenBank accession number GQ 180117) as outgroup.
Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S
rDNA gene supported strain the nearest identity for strain A4.10a (gene accession
number: KC433604) is Pseudomonas migulae, the nearest identity for strain A4.9 (gene
accession number: KC433640) is Pseudomonas mandelii, the nearest identity for strain
A4R1.12f (gene accession number: KC433636) is Pseudomonas fragi, the nearest
identity for strain A6.10 (gene accession number: KC433639) is Pseudomonas lini and
the nearest identity found for strain A5.5 (gene accession number: KC433637) is
Pseudomonas deceptionensis.
88 A4.10a
82
Pseudomonas migulae strain D67 16S ribosomal RNA gene (JN228320)
Pseudomonas gessardii gene for 16S rRNA partial sequence strain: NBRC 101045 (AB681347)
49
A6.10
72
98 Pseudomonas lini strain DLE411J (NR 029042)
Pseudomonas fluorescens strain PC17 (AY538263)(2)
95
A4.9
91 Pseudomonas mandelii strain Asd MV-11 (FM955880)
A4R1.12f
Pseudomonas fragi partial 16S rRNA gene isolate FB23 (AM933514)
100
A5.5
89 Pseudomonas deceptionensis strain M1 16S ribosomal RNA gene (GU936597)
Pseudomonas aeruginosa strain MW3A 16S ribosomal RNA gene (GQ180117)
0.005
Fig. 4.32 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Pseudomonas strains that
were isolated from Anchorage Island with closely related taxa. Bar represents
evolutionary distance as 0.01 change per nucleotide position. Bootstrap values (%) over
50% from 1,000 replications are shown.
102
4.2.12 16S rDNA gene identification and phylogenetic analysis of Arthrobacter sp.
isolated from Leonie Island
Two bacteria strains found in Leonie Island were Arthrobacter sp. The 16S
rDNA gene-based phylogenetic tree (Fig. 4.33) was rooted with Arthrobacter oxydans
strain 92-0600 (GenBank accession number EU977757) as outgroup. Phylogenetic
analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S rDNA gene
showed that the nearest identity for strain Ln5.10 (gene accession number: KC433625)
is Arthrobacter stackebrandtii and the nearest identity for strain Ln4B.1 (gene accession
number: KC433620) is Arthrobacter psychrochitini.
75 Ln5.10
86
51
Arthrobacter stackebrandtii strain : CCM 2783 16S ribosomal RNA (NR 042258)
Arthrobacter stackebrandtii strain 2P03PA (EU977757.1)
Arthrobacter psychrochitiniphilus16S rRNA strain: JCM 13874.(AB588633)
Ln4B.1
70 Arthrobacter psychrolactophilus strain KNOUC403 (HQ610444.1)
Arthrobacter oxydans strain 92-0600 (EU086811.1)
0.001
Fig. 4.33 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Arhtrobacter strains that
were isolated from Leonie Island with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
103
4.2.13 16S rDNA gene identification and phylogenetic analysis of Acinetobacter sp.
isolated from Leonie Island
Two of the bacteria strains that were found in Leonie Island is Acenitobacter sp.
The 16S rDNA gene-based phylogenetic tree (Fig. 4.34) was rooted with Arthrobacter
oxydans strain 92-0600 (GenBank accession number EU977757) as outgroup.
Phylogenetic analysis using partial nucleotide sequences (≈1.4-1.5 kbps) of the 16S
rDNA gene showed that the nearest identity for strain Ln5C.2b (gene accession number:
KC433626) is Arthrobacter psychrolactophilus and the nearest identity for strain
Ln5C.9 (gene accession number: KC433624) is Arthrobacter iwoffii.
96 Ln5C.2b
70
Acinetobacter sp. Asd MZL2 16S rRNA gene strain Asd MZL2 (FM955885)
82
Acinetobacter lwoffii strain NB11 2A 16S ribosomal RNA gene (JN644577)
Ln5.9
Acinetobacter lwoffii DSM 2403 16S ribosomal RNA (NR 026209)
Acinetobacter beijerinckii strain TY12 16S ribosomal RNA gene (JF742664)
0.001
Fig. 4.34 Phylogenetic tree constructed by neighbor-joining analysis based on the 16S
rDNA sequences depicting the phylogenetic relationship of Acinetobacter strains that
were isolated from Leonie Island with closely related taxa. Bar represents evolutionary
distance as 0.01 change per nucleotide position. Bootstrap values (%) over 50% from
1,000 replications are shown.
104
4.2.14 Screening of QQ activity of soil bacteria
For bacteria isolated from Casey Station and Rothera Research Station, the
bacteria were screened for QQ activity using CV026 overlay. 3-oxo-C6-HSL which is
best detected by CV026 was used as substrate for AHLs inactivation assay. The bacteria
isolated from Casey Station, 16 out of 21 showed positive on QQ result. For bacteria
isolated from Rothera Research Station, 31 out of 46 showed a positive QQ activity.
Fig.4.35 shows a representative data of first screening for QQ activity of Antarctic
bacteria. 3-oxo-C6-HSL after incubated with strain T7C for 24 h at 4 °C was detected
using biosensor and reduced purple pigmentation produced by CV026 overlay showed
that bacteria have inactivated the AHL.
105
4.2.15 Determination of QQ activity of soil bacteria using RRLC
After the first screening of QQ activity of Antarctic bacteria using CV026
overlay, the QQ activity of these bacteria were further confirmed with RRLC. All the
bacteria with positive results in the initial screening also showed degradations of AHLs
from RRLC analysis. However, the AHLs degradation profile was different for each
bacterial isolates. 5 bacteria from different taxa were selected to screening for all ability
to degrade different AHLs and the results were as below:
4.2.16 QQ activity of Pseudomonas spp.
QQ was found in various strains of Pseudomonas spp. in Antarctica in each soil
samples inoculated for QQ bacteria enrichment. In this study, 28 strains of
Pseudomonas spp. showed QQ activities. Pseudomonas spp. were the most abundant
QQ bacteria found in Antarctica soil sample using modified KG medium enrichment.
Below shows the representative data of QQ activity of one of the Pseudomonas sp.
using RRLC.
Fig. 4.36 shows the rate degradation of short chain AHL C4-HSL by Pseudomonas sp.
strain Ln4b.12g.
106
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-02 11-19-13\1AC-2101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-02 11-19-13\1BC-2201.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-02 11-19-13\1CC-2301.D)
mAU
160
140
0h
120
100
24 h
80
60
48h
40
20
0
0.5
1
1.5
2
2.5
3
min
3.5
0.6 min (retention time)
Fig. 4.37a RRLC analysis of synthetic C4-HSL (100µg) incubated with Pseudomonas
sp. strain Ln4b12.g at 0-, 24-, and 48-h at 4°C.
mAU
mAU
140
140
120
120
100
100
80
80
60
60
40
40
20
20
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig. 4.37b (left) and 4.37c (right) RRLC analysis of synthetic C4-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C4-HSL at 0-h
Chromatogram of C4-HSL at 24-h
Chromatogram of C4-HSL at 48-h
107
mAU
70
60
0h
50
40
24 h
30
48h
20
10
0
0.5
1
1.5
min
2
0.94 min (retention time)
Fig. 4.38a RRLC analysis of synthetic C6-HSL (100µg) incubated with Pseudomonas
sp. strain Ln4b12.g at 0-, 24-, and 48-h at 4°C Legends:
DAD1
DAD1
DAD1
DAD1
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
2011-08-09
2011-08-09
2011-08-09
2011-08-09
03-25-51\2A B -1701.D )
03-25-51\2A B -1801.D )
03-25-51\2A B -1901.D )
03-25-51\2A B -2001.D )
DAD1
DAD1
DAD1
DAD1
mAU
mAU
200
200
175
175
150
150
125
125
100
100
75
75
50
50
25
25
0
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
A , S ig=210,4
R ef=360,100
R ef=360,100
R ef=360,100
R ef=360,100
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
(C 2W S \C 2W S _C 7C A
2011-08-09
2011-08-09
2011-08-09
2011-08-09
03-25-51\2A B -1701.D )
03-25-51\2A B -1801.D )
03-25-51\2A B -1901.D )
03-25-51\2A B -2001.D )
0
0.5
1
1.5
m in
0.5
1
1.5
m in
Fig. 4.38b (left) and 4.38c (right) RRLC analysis of synthetic C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C6-HSL at 0-h
Chromatogram of C6-HSL at 24-h
Chromatogram of C6-HSL at 48-h
108
mAU
250
200
0h
150
24 h
100
48h
50
0
0.25
0.5
0.75
1
1.25
1.5
1.75
min
0.96 min (retention time)
Fig. 4.39a RRLC analysis of synthetic C12-HSL (100µg) incubated with Pseudomonas
sp. strain Ln4b12.g at 0-, 24-, and 48-h at 4°C Legends:
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 11-11-10\2AB-2501.D )
2011-08-11 11-11-10\2AB-2601.D )
2011-08-11 11-11-10\2AB-2701.D )
2011-08-11 11-11-10\2AB-2801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
mAU
mAU
50
50
40
40
30
30
20
20
10
10
0
0
0.5
1
1.5
min
0.5
2011-08-11 11-11-10\2AB-2501.D )
2011-08-11 11-11-10\2AB-2601.D )
2011-08-11 11-11-10\2AB-2701.D )
2011-08-11 11-11-10\2AB-2801.D )
1
1.5
min
Fig. 4.39b (left) and 4.39c (right) RRLC analysis of synthetic C12-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C12-HSL at 0-h
Chromatogram of C12-HSL at 24-h
Chromatogram of C12-HSL at 48-h
109
mAU
350
300
250
200
0h
150
24 h
100
50
48h
0
0.5
1
1.5
2
2.5
3
min
3.5
0.75 min (retention time)
Fig. 4.40a RRLC analysis of synthetic HHQ (10µg) incubated with Pseudomonas sp.
strain B8D at 0-, 24-, and 48-h at 4°C.
mAU
mAU
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
2
2.5
3
3.5
min
0.5
1
1.5
2
2.5
3
min
3.5
Fig. 4.40b (left) and 4.40c (right) RRLC analysis of synthetic HHQ (10µg) incubated
with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of HHQ at 0-h
Chromatogram of HHQ at 24-h
Chromatogram of HHQ at 48-h
110
Table 4.6 (a) shows the Pseudomonas strains which possess QQ activity isolated
from Browning Peninsular. Degradation of AHL: + : weak; ++ : moderate; + + + :
significant. × -no activity
Strain
B7A
B7B
B7C
B8A
B8D
C4-HSL
+++
+++
+++
+
×
C6-HSL
++
++
++
++
×
C7-HSL
++
++
+
+
×
C8-HSL
++
++
+
+
+
C10-HSL
+
+
+
+
+
C12-HSL
++
++
++
++
++
3-oxo-C6-HSL
+++
+++
+++
+
×
3-oxo-C8-HSL
++
+
+
+
++
3-oxo-C12-HSL
++
+
+
+
++
HHQ
×
×
++
×
+++
Molecules
111
Table 4.6 (b) shows the Pseudomonas strains which possess QQ activity isolated
from Thala Valley. Degradation of AHL: + : weak; ++ : moderate; + + + : significant.
× -no activity
Strain
T7A
T7B
T7C
T7D
C4-HSL
+
+
+++
+
C6-HSL
+
+
++
+
C7-HSL
+
+
+
+
C8-HSL
+
++
+
++
C10-HSL
×
×
+
+
C12-HSL
×
×
++
+
3-oxo-C6-HSL
+
+
+++
+
3-oxo-C8-HSL
+
+
+
3-oxo-C12-HSL
×
×
++
+
HHQ
×
×
×
×
Molecule
+
112
Table 4.6 (c) shows the Pseudomonas strains which possess QQ activity isolated
from ASPA 159. Degradation of AHL: + : weak; ++ : moderate; + + + : significant. × no activity
Strain
621
6PC5
6PC6
B62
B122
C4-HSL
×
+++
+
+
+
C6-HSL
+
++
+
+++
++
C7-HSL
+++
+
++
+++
++
C8-HSL
+
+
+
+++
++
C10-HSL
×
++
×
++
++
C12-HSL
×
+++
×
+++
+++
3-oxo-C6-HSL
+
+++
+
++
++
3-oxo-C8-HSL
×
+
×
+++
++
3-oxo-C12-HSL
×
+++
×
++
++
HHQ
×
×
+
×
×
Molecule
113
Table 4.6(d) shows the Pseudomonas strains which possess QQ activity for C6-HSL
that were isolated from Rothera Research Station. Degradation of AHL: + : weak;
++ : moderate; + + + : significant. × -no activity
Strain
Degradation of C6-HSL
L4R1.15 (Lagoon Island-4)
+++
L4R1.2B (Lagoon Island-4)
+
L10.9 (Lagoon Island-3)
++
L10.8 (Lagoon Island-3)
+
L10.7 (Lagoon Island-3)
++
A4R1.12f (Anchorage Island-4)
++
LN4B.7 (Leonie Island-4)
+++
LN4B.8 (Leonie Island-4)
+++
LN5C.8 (Leonie Island-5)
+++
LN4B.12g (Leonie Island-4)
+++
LN5C.7 (Leonie Island-5)
++
K3R3.1A (Killingbeck Island-3)
+
R3.1B (Rothera Point-ASPA-129)
++
R3.12(Rothera Point-ASPA-129)
+++
114
4.2.17 QQ activity of Arthrobacter sp.
Three Arthrobacter spp. that confer QQ activities were isolated from Casey
Research
Station.
These
isolates
which
closely
related
to
Arthrobacter
psychrolactophilus, Arthrobacter psychrochitinphilus and Arthrobacter stackebranalti
were found to degrade various AHL (C4-HSL, 3-oxo-C6-HSL, C7-HSL, C8-HSL, C10HSL, C12-HSL and 3-oxo-C12-HSL) at 4 °C. These three strains were isolated from
former dump site (Thala Valley) and penguin rookery (ASPA 156). However, these
bacteria were unable to degrade AHLs with carbon side chain longer than 12.
Arthrobacter sp. also found to degrade HHQ which is the signalling molecule produced
by Pseudomonas species.
Table 4.7 shows the Arthrobacter strains which possess QQ activity. Degradation of
AHL: + : weak; ++ : moderate; + + + : significant. × -no activity
Strain
Molecule
L5C.1A (Lagoon
B12C (ASPA156)
T7E(Thala Valley)
Island-5)
C4-HSL
+++
+++
+++
C6-HSL
++
++
+++
C7-HSL
+
+
+
C8-HSL
+
++
++
C10-HSL
+
+++
++
C12-HSL
++
++
++
3-oxo-C6-HSL
+++
+++
+++
3-oxo-C12-HSL
++
++
++
HHQ
++
+++
×
115
mAU
160
0h
140
120
24 h
100
48h
80
60
40
20
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Fig. 4.41a RRLC analysis of synthetic C4-HSL (100µg) with Arthrobacter sp. strain
B12C, at 0-, 24-, and 48-h at 4°C.
mAU
mAU
140
140
120
120
100
100
80
80
60
60
40
40
20
20
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig. 4.41b (left) and 4.41c (right) RRLC analysis of synthetic C4-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C4-HSL incubated at 0-h
Chromatogram of C4-HSL incubated at 24-h
Chromatogram of C4-HSL incubated at 48-h
116
mAU
250
0h
200
150
24 h
100
50
48 h
0
0.5
1
1.5
2
2.5
3
min
3.5
Fig. 4.42a RRLC analysis of samples incubated of synthetic 3-oxo-C6-HSL (100 µg)
with Arthrobacter sp. strain B12C, at 0-, 24-, and 48- hour at 4°C.
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
2011-08-09 03-25-51\2A C -3701.D )
2011-08-09 03-25-51\2A C -3801.D )
2011-08-09 03-25-51\2A C -3901.D )
2011-08-09 03-25-51\2A C -4001.D )
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
D A D 1 A , S ig=210,4 R ef=360,100 (C 2W S \C 2W S _C 7C A
mAU
m AU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
2011-08-09 03-25-51\2A C -3701.D )
2011-08-09 03-25-51\2A C -3801.D )
2011-08-09 03-25-51\2A C -3901.D )
2011-08-09 03-25-51\2A C -4001.D )
0
0.5
1
1.5
m in
0.5
1
m in
1.5
Fig. 4.42b (left) and 4.42c (right) RRLC analysis of synthetic 3-oxo-C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of 3-oxo-C6-HSL at 0-h
Chromatogram of 3-oxo-C6-HSL at 24-h
Chromatogram of 3-oxo-C6-HSL at 48-h
117
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2BA-1701.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2BB-1801.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2BC-1901.D)
mAU
350
0h
300
250
24 h
200
150
48h
100
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
min
1.8
Fig. 4.43a RRLC analysis of synthetic C12-HSL (100µg) with Arthrobacter sp. strain
B12C, at 0-, 24-, 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
2011-08-11 11-11-10\2AB-2501.D )
2011-08-11 11-11-10\2AB-2601.D )
2011-08-11 11-11-10\2AB-2701.D )
2011-08-11 11-11-10\2AB-2801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\T EST
m AU
m AU
50
50
40
40
30
30
20
20
10
10
0
0
0.5
1
1.5
m in
0.5
2011-08-11 11-11-10\2AB-2901.D )
2011-08-11 11-11-10\2AB-3001.D )
2011-08-11 11-11-10\2AB-3101.D )
2011-08-11 11-11-10\2AB-3201.D )
1
1.5
m in
Fig.4.43b (left) and 4.43c (right) RRLC analysis of synthetic C12-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C12-HSL at 0-h
Chromatogram of C12-HSL at 24-h
Chromatogram of C12-HSL at 48-h
118
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2FF-9301.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2FH-9501.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA 2011-05-12 03-48-01\2FI-9601.D)
mAU
350
0h
300
250
24 h
200
150
48h
100
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
min
1.8
Fig. 4.44a RRLC analysis of synthetic HHQ (10µg) with Arthrobacter sp. strain B12C,
at 0-, 24-, 48- h at 4°C.
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EF-8901.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EG-9001.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EH-9101.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EF-8901.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EG-9001.D)
DAD1 A, Sig=210,4 Ref=360,100 (C2WS\C2WS_C7CA2011-05-12 03-48-01\2EH-9101.D)
mAU
mAU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig.4.44b (left) and 4.44c (right) RRLC analysis of synthetic HHQ (10µg) incubated
with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of HHQ at 0-h
Chromatogram of HHQ at 24-h
Chromatogram of HHQ at 48-h
119
4.2.18 QQ Activity of Acinetobacter sp.
Two Acinetobacter spp. which possess QQ activity were isolated from Leonie
Island. These species which closely related to Acinetobacter iwoffii and Acinetobacter
beijerinckii were found to be able to degrade various AHL in 4 °C. These species were
isolated from ornithogenic under skua nest. These Acinetobacter spp. (strains Ln5C.2b
and Ln5.9) were found to be able to degrade C6-HSL, C7-HSL, C8-HSL.
Table 4.8 shows the Acinetobacter strains which possess QQ activity. Degradation of
AHL: + : weak; ++ : moderate; + + + : significant. × -no activity
Strain
Ln5C.2b
Ln5.9
3-oxo-C6-HSL
++
++
C6-HSL
++
++
C7-HSL
++
++
C8-HSL
++
++
AHLs
120
mAU
80
0h
60
24 h
40
48 h
20
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Fig. 4.45a RRLC analysis of synthetic C6-HSL (100µg) with Acinetobacter sp. strain
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3701.D )
Ln5.9,
at 0-, 24-, and 48- h at 4°C.
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3701.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3801.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -4001.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -3901.D )
D AD 1 A, Sig=210,4 R ef=360,100 (C 2W S\C 2W S_C 7C A 2011-08-09 03-25-51\2AC -4001.D )
m AU
mAU
350
350
300
300
250
250
200
200
150
150
100
100
50
50
0
0
0.5
1
1.5
m in
0.5
1
1.5
min
Fig.4.45b (left) and 4.45c (right) RRLC analysis of synthetic C6-HSL (100µg)
incubated with E.coli DH5α and PBS buffer (100mM, pH6.5) at 0-, 24-, and 48-h at 4°C.
Legends:
Chromatogram of C6-HSL at 0-h
Chromatogram of C6-HSL at 24-h
Chromatogram of C6-HSL at 48-h
121
4.2.19 Determination of homoserine lactonase activity of QQ bacteria
For bacteria isolated from Casey research station, 3 bacterial strains namely
Pseudomonas species (strain 6PC5) and two Arthrobacter spp. (strain B12C and strain
T7E) showed positive re-lactonisation of AHLs in acidic condition indicating lactonase
activity. For bacteria isolated from Rothera Research Station, eight of the bacterial
strains were found to show a significant recovery of AHLs after acidification. Four of
the bacterial strains were Pseudomonas spp. Strain L4R1.14 was isolated from Lagoon
Island, Ln5C.8 and Ln4B.12g were isolated from Leonie Island, strain A4R1.12f was
isolated from Anchorage Island. One Arthrobacter sp. strain L5C.1A was also found to
produce lactonase-type of enzyme. This strain was isolated from elephant seal wallow
from Lagoon Island. Other than Pseudomonas and Arthrobacter sp., Planococcus and
Psychrobacter sp. which have never been reported as QQ bacteria were also found to
possess homoserine lactonase activity.
122
mAU
160
140
0h
120
acidification
100
24 h
80
60
48 h
40
20
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Fig. 4.46 (a) shows the chromatogram of QQ inactivation assay for Pseudomonas sp.
strain Ln4B.12g which was isolated from Leonie Island. After acidified with HCl and
incubate for another 24 hour, a significant recovery of C4-HSL shows that strain
Ln4B.12g produce lactonase-type of enzyme to degrade AHLs.
Fig. 4.46(b) shows the recovery of concentration of C4-HSL post-acidification for
Pseudomonas sp. strain Ln4B.12g
123
Table 4.9 shows the bacterial strains that produce lactonase-type of enzyme to
degrade AHLs.
Strain
Nearest Identity
T7C
Pseudomonas sp.
T7E
Arthrobacter sp.
6PC5
Pseudomonas sp.
B12C
Arthrobacter sp.
L5C.1a
Arthrobacter sp.
L10.15
Planococcus sp.
Ln4B.12g
Pseudomonas sp.
L4R1.15
Pseudomonas sp.
A4R1.12f
Pseudomonas sp.
Ln5C.8
Pseudomonas sp.
R3.7
Psychrobacter sp.
124
4.2.20 Screening of QS activity of soil bacteria
To determine whether QQ and QS activities co-exist in Antarctic pseudomonads,
all these strains were first cross-streaked against AHL biosensors CV026 and A.
tumefaciens NTL4 (pLZR4), and the plates examined over time for the induction
production of purple pigment or degradation of X-gal which turn the biosensor into blue.
Eight Pseudomonas spp. (strains B8A, B8D, T7B, T7C, 621, 6P5, 6P6 and B12.2)
induced production of purple pigment and blue coloured colony indicating that they
produced short or long chain AHLs.
Fig.4.47 shows representative data of two of the Antarctic bacteria Pseudomonas sp.
strain L10.1 isolated from and Pseudomonas sp. strain T7B isolated from Lagoon Island
and Thala Valley (Casey Research Station), respectively. The purple pigmentation
shows the production of short chain AHLs from the two Pseudomonas spp. Equal
amount of pure ethyl acetate served as negative control.
125
Fig. 4.48 (a) The measurement of RLU/OD540 with the bioluminescent sensor strain
E.coli [pSB 401] with AHLs extract from LB broth culture of Pseudomonas sp. strain
R3.8.
.
Fig. 4.48 (b) The measurement of RLU/OD540 with the bioluminescent sensor strain
E.coli [pSB 401] with AHLs extract from LB broth culture of Pseudomonas sp. strain
D1.1.
126
Fig. 4.48 (c) The measurement of RLU/OD540 with the bioluminescent sensor strain
E.coli [pSB 401] with AHLs extract from LB broth culture of Pseudomonas sp. strain
L10.8.
Fig. 4.48 (d) The measurement of RLU/OD540 with the bioluminescent sensor strain
E.coli [pSB 401] with AHLs extract from LB broth culture of Pseudomonas sp. strain
L10.7.
127
Fig. 4.48 (a) and 4.48 (b) show the measurement of RLU/OD540 with the
bioluminescent sensor strain E.coli [pSB 401]. The increased of the reading show that
Pseudomonas sp. strain R3.8 and strain D1.1 which was isolated from ASPA 129
Donnelly Island, respectively, produced short chain AHL. Figure 4.48 (c) and 4.48 (d)
show the measurement of RLU/OD540 with the bioluminescent sensor strain E. coli
[pSB 1075]. Luminometer reading showed that Pseudomonas sp. strain L10.8 and
L10.7 produced long chain AHL. Positive control for biosensor E. coli [pSB401] was 3oxo-C6-HSL (0.001µg/µl) and for E.coli [pSB 1075] was C12-HSL (0.01µg/µl). Blank
(only biosensor without adding AHL extract) and ethyl acetate (the solvent to extract
AHLs produced by the Antarctic bacteria were used as negative control.
128
Table 4.10(a) shows the bacteria with QS activities that were isolated in Browning
Peninsular which can be detected using biosensor available. + -with activity, without activity or cannot be detected.
Strains
B7A
B7B
B7C
B7E
B8A
CV 026
+
+
-
+
+
A. tumefaciens
+
+
+
+
+
E.coli
[pSB401]
+
+
+
-
+
E.coli
[pSB1075]
+
+
+
+
+
Biosensor
NTL4 (pLZR4)
129
Table 4.10(b) shows the bacteria with QS activities that were isolated in Thala
Valley which can be detected using biosensor available. + -with activity, - without
activity or cannot be detected.
Strains
T7A
T7B
T7C
T7D
CV 026
-
+
+
-
A. tumefaciens
+
+
+
+
E.coli
[pSB401]
-
+
+
-
E.coli
[pSB1075]
-
-
+
-
Biosensor
NTL4 (pLZR4)
130
Table 4.10(c) shows the bacteria with QS activities that were isolated in ASPA 156
which can be detected using biosensor available. + -with activity, - without activity or
cannot be detected.
Strains
621
6PC5
6PC6
BC62
B112
CV 026
-
+
-
-
-
A. tumefaciens
+
+
+
+
+
E.coli
[pSB401]
+
+
+
-
+
E.coli
[pSB1075]
-
+
-
-
-
Biosensor
NTL4 (pLZR4)
131
Table 4.10(d) shows the bacteria with QS activities that were isolated in Lagoon
Island which can be detected using biosensor available. + -with activity, - without
activity or cannot be detected.
Strains
L10.7
L10.9
L10.8
L4R1.15
CV 026
-
-
+
-
A. tumefaciens
+
+
-
-
E.coli
[pSB401]
+
+
+
+
E.coli
[pSB1075]
+
+
-
-
Biosensor
NTL4 (pLZR4)
132
Table 4.10(e) shows the bacteria with QS activities that were isolated in Leonie
Island which can be detected using biosensor available. + -with activity, - without
activity or cannot be detected.
Strains
Ln5C.7
Ln5.2C
Ln5C.8
Ln4B.7
Ln4B.8
CV 026
-
+
-
-
-
A. tumefaciens
+
+
-
-
-
E.coli
[pSB401]
+
+
+
+
+
E.coli
[pSB1075]
-
-
-
-
-
Biosensor
NTL4 (pLZR4)
133
Table 4.10(f) shows the bacteria with QS activities that were isolated in Anchorage
Island which can be detected using biosensor available. + -with activity, - without
activity or cannot be detected.
Strains
A5.5
A4.10a
A6.10
A4.9
A4.6
CV 026
-
-
-
-
+
A. tumefaciens
+
+
+
-
+
E.coli
[pSB401]
+
+
+
+
+
E.coli
[pSB1075]
+
-
+
-
+
Biosensor
NTL4 (pLZR4)
134
Table 4.10(g) shows the bacteria with QS activities that were isolated in Rothera
point (ASPA 129), Donnelly Island and Killingbeck Island which can be detected
using biosensor available. + -with activity, - without activity or cannot be detected.
Strains
Biosensor
D1.1
K3R3.2
K3.4
( Donnelly) (killingbeck) (Killingbeck)
R3.8
R3.9
(Rothera
(Rothera
Point)
Point)
CV 026
+
-
-
-
-
A. tumefaciens
+
-
-
-
-
E.coli
[pSB401]
+
+
+
-
+
E.coli
[pSB1075]
-
-
-
-
-
NTL4
(pLZR4)
135
4.2.21 Characterization of AHLs and HHQ produced by Antarctic soil bacteria
using TLC and LC/MS/MS
To further identify the type of AHLs produced, AHLs extracted from these
bacterial strains (strains B8A, B8D, T7B, T7C, 621, 6P5, 6P6 and B12.2) were analyzed
by high resolution LC/MS. From the LC/MS analysis, all the strains except BC62 and
B122 produced unsubstituted C12-HSL. Fig. 4.52b shows the fragmentation patterns for
C12-HSL (precursor ion m/z 95, 102, 109 [M+H] and fragment ions m/z 284 produced
by strain T7C. Strain T7C also produced C16-HSL (m/z 340). From LC/MS analysis
BC62 and B122 were produce C16-oxo-HSL m/z 354. Two strains 6121 and 6PC5 were
producing a rare odd number carbon side chain long chain AHL, C15-oxo-HSL (m/z
340;). The mass spectra of the extracted AHLs were similar to the corresponding
synthetic compounds.
Fig.4.49 (a) shows representative data of TLC which indicates the QS activity of 2
Antarctic Pseudomonas strains using CV026. Fig. 4.21 (b) shows representative data of
TLC which indicates the QS activity of 3 Antarctic Pseudomonas strains using A.
136
tumefaciens NTL4 (pLZR4). The blue spot revealed the position of AHLs. Synthetic
C12-HSL was used as positive control.
Fig. 4.49 (b) Representative data of TLC which indicates the QS activity of 3 Antarctic
Pseudomonas strains using A. tumefaciens NTL4 (pLZR4).
Fig. 4.50 shows representative data of QS activity for 3 out of 11 Antarctic strains
using A. tumefaciens NTL4 (pLZR4). The blue spot revealed the position of AHLs.
Synthetic AHLs were used as positive control.
137
PQS
PQS
HHQ
HHQ
A5R1.9a
R3.7
Fig. 4.51 shows the TLC chromatographed of organic solvent extract from bacterial
strain A5R1.9a isolated from Anchorage Island (first lane from left) and strain R3.7 that
were isolated from Rothera point –ASPA 129 (second lane from left). Third lane from
left is standard HHQ and forth lane from left is standard PQS. Strain A5.9 was found
producing both HHQ and PQS, however strain R3.7 only was found to produce HHQ.
According to TLC result, among Pseudomonas spp. that we were isolated from
Casey Research Station, 1 out of 14 produced HHQ, however, none of these strains
produced PQS. For Pseudomonas spp. that were isolated from Rothera Research Station,
3 out of 20 were producing HHQ, 1 out of the 20 Pseudomonas spp. produced PQS.
Psychrobacter cryohalolentis strain R3.7 which is close relative with Pseudomonas sp.
(same class) was found to produce HHQ as well.
138
Table 4.11 shows the bacteria that produce HHQ or PQS based on thin layer
chromatography.
+ detected, - not produce or cannot be detected
Strain
Nearest Identity
Production of HHQ
Production of PQS
62.1
Pseudomonas sp.
+
-
A5.9
Pseudomonas sp.
+
+
R3.7
Psychrobacter sp.
+
+
Ln5.2c
Pseudomonas sp.
+
-
D1.1
Pseudomonas sp.
+
-
139
Fig. 4.52 (a) shows the spectrum of the organic solvent extract of strain T7C. The arrow
shows the spectrum of the AHL 3-oxo-C8-HSL (peak m/z 242.14). Fig. 4.52 (b) shows
the spectrum of LCMS/MS analysis of peak m/z 242.14 (3-oxo-C8-HSL), the peaks
show the product ions of 3-oxo-C8-HSL, m/z 102.0547 and 141.0904.
140
Fig.4.52 (b) The spectrum shows a representative result of LCMS analysis from
bacteria strain B7A. The spectrum above showed that the bacteria produced C12-HSL
(m/z 284.2197). Strain B7A was also found to produce short chain AHL 3-oxo-C8-HSL
(m/z 242.14) as well.
Table 4.12 shows the AHLs profile from bacteria that were isolated from Casey
Research Station. The AHLs extracted from the spent supernatants were assayed using
LCMS/MS analysis.
Isolates
B7A
Bacteria identity
Pseudomonas brenneli
HSL and Oxo-HSL
Location
3-oxo-C8-HSL( m/z 242.14; 4 Browning
Peninsular
min; m/z 102.0547 ,141.0904)
C12-HSL (284.2197; 5.81min;
m/z 95, 109)
B7B
Pseudomonas
frederiksbergenensis
C12-HSL
(
m/z
284.2192; Browning
Peninsular
5.94min; m/z 95, 109)
141
C16-HSL
(
m/z
340.2815;
(
m/z
284.2198; Browning
Peninsular
9.16min)
B7C
Pseudomonas migulae
C12-HSL
5.89min; m/z 95, 109)
C16-HSL
(
m/z
340.2821;
(
m/z
284.2197; Browning
Peninsular
(
m/z
340.2801;
(
m/z
284.2212; Browning
Peninsular
9.13min)
B8A
Pseudomonas lini
C12-HSL
5.91min)
C16-HSL
9.13min)
B7E
Pseudomonas gessardii
C12-HSL
5.92min; m/z 95, 109)
C16-HSL
(
m/z
340.2826;
9.19min)
T7A
Pseudomonas poae
C8-HSL( m/z 228.16; 4.7 min;
Thala
Valley
m/z 102.0560 ,210.1478)
C12-HSL
(
m/z
284.2198;
5.89min; m/z 95, 109)
C16-HSL
(
m/z
340.2829;
(
m/z
284.2186; Thala
Valley
9.13min)
T7B
Pseudomonas antarctica
C12-HSL
5.89min)
T7C
Pseudomonas fluoresces
C8-HSL( m/z 228.16; 4.7 min;
Thala
Valley
m/z 102.0560 ,210.1478)
3-oxo-C8-HSL( m/z 242.14; 4
142
min; m/z 102.0547 ,141.0904)
C12-HSL
(
m/z
284.2203;
5.68min; m/z 95, 102, 109)
C16-HSL
(
m/z
340.2811;
(
m/z
284.2220; Thala
Valley
9.13min)
T7D
Pseudomonas
frederiksbergenensis
C12-HSL
5.91min; m/z 95, 109)
C16-HSL
(
m/z
340.2815;
(
m/z
284.2236; ASPA 136
9.13min)
62.1
Pseudomonas migulae
C12-HSL
6.87min; m/z 95, 109)
C16-HSL
(
m/z
340.2823;
10.32min)
3-C15-Oxo-HSL
(
m/z
340.2475; 10.06min)
6PC5
Pseudomonas antarctica
C12-HSL
(
m/z
284.2232; ASPA 136
6.92min; m/z 95, 102, 109)
C16-HSL
(
m/z
340.2874;
10.33min)
C15-oxo-HSL ( m/z 340.2494;
10.12min)
6PC2
Pseudomonas
psychrophila
C12-HSL
(
m/z
284.2206; ASPA 136
6.98min; m/z 95, 102, 109)
143
BC62
Pseudomonas syringae
C16-oxo-HSL ( m/z 354.2639; ASPA 136
9.97min)
B12.2
Pseudomonas mendelii
C16-oxo-HSL ( m/z 354.2639; ASPA 136
9.97min)
144
CHAPTER 5
DISCUSSION
5.1 Isolation of AHL degrading bacteria
From the enrichment of using KG medium supplemented with C12-HSL as sole
source of carbon and nitrogen, 11 Antarctic pseudomonads and 3 Arthrobater spp. were
isolated and all the bacteria grew well at 4°C. These strains are actually psychrotolerant
and able to grow from 4-30°C. This demonstrated that our previously reported KG
medium is useful to enrich bacteria from this pristine environment of Antartica. When
KG medium (supplemented with 3-oxo-C6-HSL as energy soruce) was inoculated with
tropical soil, a variety of QQ bacteria were isolated such as Acinetobacter, Burkholderia
and Klebsiella (Chan et al., 2011). However, when inoculated with Casey station’s soil
sample, only Pseudomonas spp. and Arthrobacter spp. were enriched.
Different
profiles of bacteria enriched by KG medium could be due to (1) the use of C12-HSL as
sole carbon and nitrogen source in this study and, (2) different soil samples used, (3)
growth temperature of 4°C for 21 days, (4) the soil sample was not inoculated
immediately after collection.
For the enrichment which was done in Rothera Research Station and the isle
nearby, fresh soil were used. Although Pseudomonas sp. strains and Arthobacter sp.
strain with QQ activity were mainly isolated the attempt has managed to isolate a few
novel QQ bacteria including Psychrobacter sp. strain R3.7, Planococcus sp. strain
L10.15 and Flavobacterium sp. strain Ln5b.12. Psychrobacter sp. strain R3.7 was
isolated in Antarctic Special Protected Area. A well studied psychrotolerant strain
Psychrobacter cryohalolentis strain K5 was isolated from a cryopeg (saline water lens)
within 40 thousand-year-old Siberian permafrost where the in situ temperature was -9 to
145
-11°C (Bakerman et al., 2003). This strain is very important for researchers to study
evolution and response of bacteria when exposed to extremely low temperatures such as
the implications of subzero metabolic activity on long-term microbial survival in
terrestrial and extraterrestrial permafrost (Amato et al., 2010). Planococcus sp. strain
L10.15 was isolated from elephant seal wallow from Lagoon Island. Planococcus sp.
was reported to degrade compounds with benzene ring in multiple extreme conditions.
(Li et al., 2006). Flavobacterium sp. strain Ln5b.12 was isolated from soil under skua’s
nest from Leonie Island and this strain also was found to produce QS signal as well.
Flavobacterium spp. are generally opportunistic pathogens that live in soil and water,
for example Flavobacterium psychrophilum, a pathogen that causes a severe diseases in
fish, especially in farmed rainbow trout (Oncorhynchus mykiss) (Garcia et al., 2000).
Antarctic bacteria isolates showed broad QQ activities with broad specificity as
they are able to degrade wide range of AHLs. Interestingly, all these QQ bacteria
isolates found in Casey station produced C12-HSL, suggesting QS activity, but only
strain T7A (Pseudomonas poae), T7B (Pseudomonas antarctica) and strain
621(Pseudomonas migulae) were not able to degrade the C12-HSL which produce by
itself. Strain T7A, strain T7B and strain 621 significantly degraded short chain AHLs
and showed only minimal C12-HSL degradation. As such, Antarctica pseudomonads
were shown to produce and degrade the AHLs produced, for example C12-HSL. Such
finding may imply that Antarctic pseudomonads finely regulate their production of
AHLs whereby once the QS-dependent phenotypes are accomplished, rapid turnover of
AHLs were observed.
146
Another three strains which were isolated from Antarctica with QQ abilities are
Arthrobacter spp. (Strain L5C.1A, B12C and T7E). Arthrobacter spp. isolated from
temperate soil sample were also found to degrade AHLs ranging from C4 to C12-HSL
(Park et al., 2003). In addition, this is the first reported Antarctic Arthrobacter strain
with QQ ability.
Eventhough various AHLs were used as carbon source for enrichment medium,
which aim to enrich and isolate different bacteria from selected soil samples, however,
bacteria from same genus (example: Pseudomonas spp.) were isolated from all the
selected soil samples. This suggested that different AHLs as carbon source in KG
medium has no effect for bacteria isolation.
5.2 Discovery of novel QQ bacteria
Bacterial diversity in Antarctica is very different from any other places in the
world as it harbors one of the most environmentally harsh ecosystems. The diversity
was always affected by a lot of factors especially temperature. Cold-adapted
psychrotolerant and psychrophile bacteria are always the prevalent in the Antarctic
ecology system. The soil bacterial diversity which have been affected or limited by
carbon source (Wynn-william 1990) is poorly studied. Therefore, using AHL as carbon
source to QQ bacteria can extend our knowledge to new QQ bacteria that have never
been isolated in other part of the world.
Psychrobacter sp. strain R3.7 which is close to Psychrobacter cryohalalentis
(99% in NCBI Blast) is the first novel QQ bacterium that was isolated from Rothera
Research Staion (ASPA 129). The closest genus with QQ activity for tropical strain will
147
be Pseudomonas sp. (γ-Proteobacteria). Pseudomonas spp. with QQ abilities were also
found in several sites of Antarctica. Psychrobacter spp. were found in different
locations and samples collected from different environments of Antarctica including
ornithogenic soil (Bowman et al., 1996), Antarctic sponge (Papaleo et al., 2011), icesealed lake (Mondino et al., 2009), Antarctic krill (Denner et al., 2001), pack ice
(Brinkmeyer et al., 2003) and fast ice (Shivaji et al., 2004). Psychrobacter sp. isolated
from Antarctic sponge species was found to possess antimicrobial ability toward
Burkholderia cepacia complex (Bcc).
In present, Planococcus sp. is the only novel Gram positive bacteria being
isolated in Antarctica by this present study. The nearest mesophilic relative that
possesses QQ activity is Bacillus sp. Planococcus sp. strain L10.15 possessed same
ability as Bacillus sp. which produced AHL lactonase enzyme that cleaves the ester
bond of the homoserine lactone ring (Dong et al., 2000). Planococcus spp. were found
in several location in Antarctica, including soil from seal wallow (Chong et al., 2009a),
Sub-Antarctic sea ice (Olivera et al., 2007), Antarctic soil sample (Miller et al., 1983).
Flavobacterium sp. strain Ln5B.12 is the second member of Bacteriodetes
which possess both QS and QQ activity, the previous reported member with QQ
abilities was a fish pathogen Tenacibaculum maritimum (Romero et al., 2010).
Therefore, the discovery of Flavobacterium sp. strain Ln5B.12 with QQ abilities in
Antarctica has extends the paradigm of AHL-mediated QS signalling beyond the
Proteobacteria and reinforces its ecological significance. Flavobacterium spp. that were
isolated in this work showed both QS and QQ activities, but the mechanism of AHL
degradation has yet to be confirmed.
148
To determine the mechanism of AHL inactivation by Psychrobacter sp. strain
R3.7, and Planococcus sp. strain L10.15,
RRLC analysis was performed and it was
suggested that these two bacteria inactivated AHLs via lactonase activity. However
bacteria may possess more than one QQ mechanism, for example Rhodococcus
erthropolis strain W2 was found to be able to inactivate AHLs via lactonase, acylase
and oxidoreductase activities (Uroz et al., 2003, Uroz et al., 2005, Park et al., 2006,
Uroz et al., 2008).
From the AHL inactivation profile of Flavobacterium sp., that selectively
inactivates short chain AHLs, it may give a competitive advantage against other short
chain producing QS bacteria. However, the mechanism on how this bacteria to degrade
short chain AHLs remains unknown. The AHLs were found not to recover after
acidification with HCl. This indicates that inactivation of AHLs by this bacteria is not
lactonase-type of enzyme.
5.3 Kinetic analysis of AHL turnover from novel QQ bacteria
Since no reported work has been done to examine the kinetic of psychrophilic
Antarctic bacterial strains on AHL turn over, this work has set to study the enzymatic
AHL-degradation of the three novel QQ strains using RRLC. From the standard curves
constructed using synthetic AHL standards, it was found that the amount of AHL
degraded is directly proportional to the areas below the curve of the RRLC
chromatograms within the working range of 0.5 to 2.0 µg/µl (R2 >0.99). Hence, using
linear regression analysis, residual AHL concentration at each time interval can be
determined through interpolation. By plotting the residual AHL concentrations against
time on a semi-log scale, the AHL degradation kinetic can be determined from the
linear portion of the curve.
149
For Psychrobacter sp. strain R3.7, it was found that this isolate rapidly degraded
all the AHLs tested. This isolate was found to favorably degrade AHLs with 8 carbon
side chain, which includes C8-HSL, and 3-hydroxy-C8-HSL. A 100 µl of resuspended
resting cells (OD600 1.0) can degrade 100 µg of both synthetic AHLs in 48 h at 4 °C (≈
30 ng h-1 per 109 CFU/ml). As compared to bacteria strains found in tropical countries
such as Bacillus cereus which rapidly degrade 3-oxo-C6-HSL and 3-oxo-C8-HSL (4.98
and 6.56 µg h-1 per 109 CFU/ml) (Chan et al., 2010), the kinetic is relatively low,
however, the Antarctic strains was able to work at 4º C This strain also degrades all the
AHLs tested. The degradation rate varied but the rate for C5-HSL, C6-HSL, 3-oxo-C6HSL and 3-hydroxyl-C6-HSL were relatively slow.
Planococcus sp. strain L10.15 can degrade AHLs with carbon side-chain up to
12. , this strain did not degrade C12-HSL. C12-HSL was found to be produced by a lot
of Pseudomonas spp. in around Casey and Rothera station which characterized by
LC/MS/MS. This strain also degrades C4-HSL at very high rate (50 ng h-1 per 109
CFU/ml).
Flavobacterium sp. strain Ln5b.12 was found not to degrade long chain AHLs.
This strain only degrades AHLs with carbon side chain up to 10. Similar to
Psychorobacter sp. strain R3.7, this strain preferably degrades AHLs with medium side
compare to other AHLs. This strain was found most effectively degrade C7-HSL, C8HSL, and 3-hydroxyl-C8-HSL (≈ 20 ng h-1 per 109 CFU/ml). Other than that, this
bacteria was found to effectively degrade C4-HSL which same with other two novel QQ
bacteria (Psychrobacter sp. strain R3.7 and Planococcus sp. strain L10.15).
150
5.4 QS activity of QQ psychrotolerant bacteria
Various Pseudomonas spp. were isolated from ecologically different sites of
Antarctica, including Pseudomonas antarctica, Pseudomonas syringae, Pseudomonas
fragi,
Pseudomonas
corrouta,
frederiksbergenensis, Pseudomonas
Pseudomonas
brenneli,
migulae, Pseudomonas
Pseudomonas
lini, Pseudomonas
gessardii, Pseudomonas poae, Pseudomonas fluoresces, Pseudomonas psychrophila
and Pseudomonas mendelii.
Pseudomonas spp. that we isolated from these sites possess ability to produce
AHLs, some of the Pseudomonas spp. were showed a strong positive result for
biosensor CV026 suggesting production of short chain AHLs. Pseudomonas spp. with
positive result in CV026 were further characterized with normal phase thin layer
chromatography. From the results, Pseudomonas spp. produced C4-, C6- and C8-HSL.
However, from the amount of the purple pigments produced by CV026, all these strain
produce different amount of AHLs.
For biosensor Agrobacterium tumefaciens NTL4 (pZLR4), a broad range
biosensor that detect AHLs with acyl-chain from C4- to C12-HSL, showed that the
Pseudomonas spp. from Antarctica produce both unsubstituted and oxo group of AHLs.
However, we could not characterize all the AHLs produced by these Antarctic
Pseudomonas due to lack of AHL standard especially AHLs with hydroxy side chain.
151
5.5 Characterization of signaling molecules produced by psychrotolerant bacteria
From LCMS/MS analysis, pseudomonads from Casey Station produced C12HSL (Table 4.7). However, some pseudomonad strains, for example Pseudomonas lini,
did not produce any detectable AHLs including analysis using LCMS/MS. Among the
strains with positive results on the biosensor, pseudomonads from Casey station were
found to produce various long chain AHLs.
To this date, no pseudomonads that produce C12-HSL were reported except for
3-oxo-C12-HSL (Steidle et al., 2002, Wagner et al., 2003). 3-oxo-C12-HSL is
important for biofilm development in pseudomonad strains such as plant saprophyte P.
putida (Steidle et al., 2002). However, this work showed that close species of Antarctic
P. putida (stain T7B) isolated from Thila Valley produced only C12-HSL. P.
aeruginosa uses 3-oxo-C12-HSL for production of virulence factor and regulation of
another signalling molecule (4-quinolone signalling). C12-HSL was found in Brucella
melintensis, and was up-regulating expression of antibiotic and toxin resistance gene,
stress survival aids, energy production genes (Weeks et al., 2010). However, the
functional studies of this signalling molecules in Antarctic pseudomonads were not
included in this studied, therefore the function of this C12-HSL remain unknown.
Among all the bacteria isolates, only Pseudomonas sp. stain T7C was confirmed
to produce C6-HSL as detected by LCMS/MS. The production of C6-HSL was found in
biocontrol strain P. chlororapis and was important in production of phenazine-1carboxamide (Chin et al., 2001, Chin et al., 2005). It has also been reported that C6HSL regulates gene activities for phenazine antibiotic synthesis in P. aureofacines and
P. aeruginosa (Pierson et al., 1994, Wood et al., 1997, Zhang & Pierson 2001).
152
Therefore, more study should be carried out in Pseudomonas sp. isolated from
Antarctica which may produce phenazine.
Most of the bacteria isolated from KG medium produced C12-HSL during the
exponential phase (data not shown). The role of this C12-HSL produced by
psychrophilic Antarctic pseudomonads remain unknown, however, C12-HSL also found
to be produced by Brucella melitensis and has been shown to regulate expression of a
large and diverse number of genes hence suggesting an important contribution to
intracellular survival (Weeks et al., 2010). Acidithiobacillus ferrooxidans was also
reported to produce C12-HSL (Farah et al., 2005). The actual function of C12-HSL in
this extremophile is unknown, but recently, Wenbin et al., 2011 demonstrated that by
using synthetic QS blocker, (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone, the
bacteria seemed to change the gene expressions for Cu2+ resistance.
P. aureofaciens isolated from root hair of sugar beet plant was reported to
produce 3-hydroxy-C14:1-HSL, C10-HSL and C6-HSL which regulate the production of
mupirocin (Laue et al., 2000). In the phytopathogen P. syringae pv. syringae produce
3-oxo-C6-HSL to regulate exopolysaccharide, swimming motility and virulence of the
bacteria (Quinones et al, 2004, Quoinones et al., 2005).
The AHL profiles for Pseudomonas spp. that were reported on environmental or
clinical isolates were different from those of the Antarctic isolates. It is evident that
C12-HSL is predominantly produced by Antarctic pseudomonads. Some Pseudomonas
spp. that were isolated in Antarctica are close to P. antarctica, P. fragi, P. coruta, P.
153
marginalis, P. brenelli which were never been reported to produce AHLs. However, 3oxo-C12-HSL which is encoded by lasI synthase was not detected in the extracted
supernatant of Antarctic pseudomonads. Since Antarctic pseudomonads commonly
produced C12-HSL, this AHL could be important for Pseudomonas spp. to regulate
gene expression in this pristine environment.
Other long chain AHLs such as C16-HSL, C16-oxo-HSL and C15-oxo-HSL
were also detected by high resolution LC/MS. Bacterial species including
Agrobacterium vitis, Paracoccus denitrificans, Rhodobacter capsulatus, Rhizobium
leguminosarum, and Sinorhizobium meliloti have been shown to produce AHLs
containing acyl side chains longer than 12 carbons (Hao et al., 2006, Llamas et al., 2004,
Schaefer et al., 2002). C16-HSL produce by Agrobacterium vitis, was found encoded by
avsI (Savka et al., 2011). C16-HSL was found to activate gene exchange between R.
capsulatus cells (Llamas et al., 2004). Ortori et al., 2011 has profiled the AHLs
production from P. aeruginosa PAO1 and has found production of 3-oxo-C16-HSL. A
fish pathogen Pseudomonas sp. strain W3.1 also found to produce 3-oxo-C16-HSL
(Chan et al., 2012). However, the functions of 3-oxo-C16-HSL in Pseudomonas sp.
remain unknown.
For Antarctic pseudomonads strains, we found a few strains that produce these
long chain AHLs. Those strain produce long chain AHLs in early stationary phase and
were detected by LC-MS. P.s borealis were found to produce C12-HSL, 3-oxo-C15HSL and C16-HSL, but 3-oxo-C15-HSL and C16-HSL were produced in trace amount.
The functions of these AHLs remain unknown for those Antarctic strains.
154
5.6 Important of discovery of QQ and QS Antarctic bacteria
The observation of QQ for Antarctic bacteria is striking since these suggest that
the Antarctic microbiota have invested in enzymes to accelerate the degradation of AHL
molecules. These bacteria are able to inactivate both short and long chain AHLs
regardless of its unsubstituted or with oxo- or hydroxyl- side chain. In this study, shortchain AHLs degraded more rapidly than long-chain AHLs. The results were similar to
the studied conducted by Yates et al., 2002, however, Yates et al., 2002, conducted the
studied in tropical ambient temperature. The recyclization of degraded AHLs lactones
ring showed that Psychrobacter sp. strain R3.7 and Planococcus sp. L10.15 produces
lactonase-type enzyme that hydrolyzes the ester bond of the lactone ring. This is also
the first reported Antarctic strains that possess QQ activity. Compared to the bacteria
strains that were isolated from tropical and temperate regions, Antarctic bacteria with
QQ activity can effectively degrade AHLs at as low as 4°C.
QS confers a strong competitive advantage among the relevant microbes living
in diverse habitats. Antarctica is the most challenging habitat on Earth; the coldest,
driest, highest (on average) and windiest continent. For bacteria, working in a
population density-dependant manner will be more effective than only a single bacterial
cell. This phenomenon, where a bacterial population behaves co-operatively and
recognizes self from non-self, could be highly advantageous particularly in the contexts
of symbiosis and niche adaptation, production of secondary metabolites, and for
facilitating population migration if prevailing condition in a specific environment niche
have become unfavourable.
155
Prokaryotes dominate many Antarctic ecosystems and play major roles in food
chain, biogeochemical cycles and the mineralization of pollutants. From the biodiversity
studies from different locations in Antarctica, for example Signy Island (Chong et al.,
2009b), showed a high proportion of γ-proteobacteria ie. Acinetobacter spp. and
Pseudomonas spp. The mesophilic strains of all these bacteria have been reported to
use AHLs as their QS signal (Kang et al., 2004, Huang et al., 2003). Therefore,
inactivation of AHLs by P. cryohalolentis strain may be an important survival skill in
Antarctica with reference to its ability to compete with QS bacteria, and the ability to
derive energy from AHLs.
As reported by Gram et al., 1999 and Bruhn et al., 2004, AHLs were found in
spoilt food and many Gram negative, AHLs-producing bacteria were isolated from the
spoilt food. Bruhn et al., 2004 has demonstrated that synthetic QS inhibitor,
halogenated furanones C-30 which added to the freezing meat samples make no
different between the non added preserve foods. However, Hentzer et al., 2003 showed
that biofilms grown with or without furanones C-30 were not significantly different in
biofilm formation and biomass. Halogenated furanones C-30 also exhibits both toxicity
and possible carcinogenic properties which unsuitable for human usage (Rasch et al.,
2004). On the other hand, the vacuum-packed cold-smoked salmon has found to contain
AHLs which produced by Enterobacteriaceae, even though the function of the AHLs
were remain unknown in the food samples (Gram et al., 1999) Hence, the QQ enzymes
produce by these psychrophilic bacteria which able to inactivate AHLs in low
temperature were potentially important in food preservation.
156
Both Arthrobacter psychrolactophilus strains were reported as non-pathogenic
cold-adapted bacteria which is able to produce exoenzymes at low temperature (Smith
& Zahnley 2005). Enzymes which have maximal activity at low temperatures have
greater specific activities at low temperatures than enzymes with higher temperature
(Feller et al., 1994). This could reduce costs for enzymes used in low-temperature
applications. Therefore, the ability of these non-pathogenic strains is potentially
important for industrial purposes. The strains that we found in Antarctica were produce
lactonase enzyme similar to previous reported tropical bacteria strain such as
Arthrobacter sp (Park et al., 2003). Hence, this cold-adapted enzyme has high potential
to replace other antibacterial agents.
5.7 Future work
The major finding of this study is the identification of Antarctic QQ and QS
bacteria. These including three novel QQ bacteria Psychrobacter sp. strain R3.7,
Planococcus sp. strain L10.15 and Flavobacterium sp. Ln5b.12. Many intriguing
questions were rised from the identification of these novel QQ bacteria including: What
is the exact chemistry and metabolic pathway of the AHL-degradation? What is the
ecological role of the AHL-degrading mechanism in Antarctica strains and the potential
use of the AHL-degradation mechanism in low temperature?
P. cryhololentis for example, a genome study of this P. cryohalolentis strain K5
showed that this strain possess 3-oxoadipate enol lactonase gene, another lactonase type
of enzyme but not AHL lactonase. Therefore, looking for gene regulation of all these
157
species may lead us to a better understanding of psychrophilic or psychrotolerant
bacteria.
Another interesting point is that QS activities possess by Antarctic bacteria
especially Antarctic pseudomonads. The AHL profile for Antarctic pseudomonads is so
different with mesophilic pseudomonads. Studies on the difference in AHL profile may
lead us to understanding the roles of these AHLs produced by these bacteria. The
question on whether AHLs may regulate expression cold tolerant protein or their
survivability in this pristine environment is of particular interest.
Future work may be focused on these questions that extensive study on the QQ
and QS systems in psychrophilic bacteria lead us to a better understanding of the
microbial ecology as well as QS-mediated gene expression of these bacteria that live in
extreme environment.
158
Conclusions
The present work demonstrated for the first time the QS and QQ activities from
bacteria that were isolated from Antarctica. The findings of QS and QQ activities of
bacteria in low temperature (4°C) show that bacteria cell-to-cell communication may
play an important role for the bacteria in this extreme environment to survive or
compete with each other. This finding may be important to delve deeply the ecosystem
in Antarctica, as prokaryote is the major component in most of the habitats of this
region.
A few novel QQ bacteria including Planococcus sp., Flavobacterium sp. and
Psychrobacteria sp. were isolated from different locations of Antarctica. However, most
of the bacteria that were found to possess QS or QQ activity were similar with
temperate region. Those bacteria including Pseudomonas sp., Arthrobacter sp. and
Acinetobacter sp. which were also found in Antarctica, however, the QS and QQ
activities were found active in low temperature (4°C).
The LC/MS studied shows that Pseudomonas sp. were produced different AHLs
including the rare produced AHLs, C15-HSL and 3-oxo-C15-HSL. The bacteria also
were found to produce HHQ, a quinolone signaling molecule in low temperature (4°C).
Bacteria such as Psychrobacter sp., Planococcus sp., Pseudomonas sp. and
Arthrobacter sp. were also found to degrade the signaling molecules, AHLs, with
homoserine lactonase type of enzyme. Interestingly, Planococcus sp. was found able to
degrade HHQ as well.
More investigation is needed to gain further insights into the ecological role and
the gene level regulation of these mechanisms. The roles of QS and QQ activity may
hold great promises in the understading of Antacrtic ecosystem or extreme environment.
159
Appendix I Gene Accession Number of Isolated Bacteria
Sampling site
Thala Valley
Bacteria
strain
T7A
Pseudomonas sp.
16S rDNA sequence
(Gene ref. number)
KC479345
66º16’49’’S;
T7B
Pseudomonas sp.
KC433633
110º32’14”E
T7C
Pseudomonas sp.
KC433634
T7D
Pseudomonas sp.
KC433635
T7E
Arthrobacter sp.
KC433651
B7A
Pseudomonas sp.
KC433605
B7B
Pseudomonas sp.
KC433652
B7C
Pseudomonas sp.
KC433606
B7E
Pseudomonas sp.
KC433647
B8A
Pseudomonas sp.
KC433602
B8D
Pseudomonas sp.
KC433607
Browning
Peninsula
Bacteria Identity
66º28’20”S;
110º32’59”E
Antarctic Special
Protected Area
136
621
Pseudomonas sp.
KC433600
B62
Pseudomonas sp.
KC433608
66º15’08’’S;
6PC5
Pseudomonas sp.
KC433603
110º32’15”E
6PC2
Pseudomonas sp.
KC433599
B122
Pseudomonas sp.
KC433656
B12C
Arthrobacter sp.
KC433612
Lagoon Island
L1R3.5
Pseudomonas sp.
KC433653
S 67° 35.689'
L1R3.6
Pseudomonas sp.
KC433616
W068° 14.495'
L10.15
Planococcus sp.
KC479344
160
Lagoon Island
L10.1
Pseudomonas sp.
KC433614
S 67° 35.613'
L10.4
Pseudomonas sp.
KC505201
W068° 14.960'
L10.10
Pseudomonas sp.
KC433618
L5C.1A
Arthrobacter sp.
KC433655
Leonie Island
Ln4B.1
Arthrobacter sp.
KC433620
S 67° 35.607
Ln4B.7
Pseudomonas sp.
KC433641
Ln4B.8
Pseudomonas sp.
KC433642
Ln4B.12g
Pseudomonas sp.
KC433644
Ln5C.2B
Acinetobacter sp.
KC433626
Ln5C.7
Pseudomonas sp.
KC433645
Ln5B.12
Flavobacterium sp.
KC433611
Ln5.9
Acinetobacter sp.
KC433624
Ln5.10
Arthrobacter sp.
KC433625
Ln5C.8
Pseudomonas sp.
KC433643
R3.1B
Pseudomonas sp.
KC433649
R3.7
Psychrobacter sp.
KC433615
R3.8
Pseudomonas sp.
KC433630
R3.9
Pseudomonas sp.
KC433632
R3.12
Pseudomonas sp.
KC433650
D1.1
Psychrobacter sp.
KC433610
K3.4
Pseudomonas sp.
KC433648
W068° 20.670'
Leonie Island
S 67° 35.627'
W068° 20.675'
Rothera Point
S 67° 34.102'
W068° 06.852'
Donelly Island
S 67° 36.153'
W068° 12.156'
Killingbeck
161
Island
K3R3.1A
Pseudomonas sp.
KC433646
K3R3.2
Pseudomonas sp.
KC433622
A4.9
Pseudomonas sp.
KC433640
A4.10a
Pseudomonas sp.
KC433604
A4R1.12f
Pseudomonas sp.
KC433636
A5.5
Pseudomonas sp.
KC433637
A6.10
Pseudomonas sp.
KC433639
S 67° 35.312
W068° 07.156'
Anchorage
Island
S 67° 36.813'
W068° 12.551'
Anchorage
Island
S 67° 36.223'
W068° 12.447'
Anchorage
Island
S 67° 36.160'
W068° 12.455'
162
Appendix II Standard curves
RRLC analysis of synthetic standards of 2-heptyl-4-quinolone (HHQ)
mAU
1000
800
600
400
200
0
0
0.5
1
1.5
2
min
2.5
Retention time: 0.85 min
Report of calibration curve for HHQ
Compound #1, VWD1 A
Area = 3.32854776*Amt +16.060524
Are
350
Rel. Res%(1): 1.224
5
300
250
4
2000
3
2
150
100
1
50
0
Correlation: 0.99996
0
50
Amount[ng/ul
163
RRLC analysis of synthetic standards of synthetic C4-HSL
mAU
250
200
150
100
50
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Retention time: 0.60 min
Report of calibration curve for C4-HSL
Compound #1,
Area = 0.43402968*Amt +45.6716
Area
Rel. Res% (1): 17.415
4
800
600
2
400
3
1
200
0
Correlation: 0.99432
0
1000
Amount[ng/ul]
164
RRLC analysis of synthetic standards of synthetic C6-HSL
mAU
250
200
150
100
50
0
0.5
1
1.5
2
min
2.5
Retention time: 1.0 min
Report calibration curve of C6-HSL
Compound #1,
Area = 0.54760225*Amt +5.9835815
Area
Rel. Res% (1): 1.833
1000
4
800
2
600
400
3
1
200
0
Correlation: 0.99994
0
1000
Amount[ng/ul]
165
RRLC analysis of synthetic standards of synthetic 3-hydroxy-C6-HSL
mAU
120
100
80
60
40
20
0
0.2
0.4
0.6
0.8
1
1.2
min
1.4
Retention time: 0.62 min
Report calibration curve of 3-hydroxy-C6-HSL
Com pound #1,
Area = 0.23644279*Am t +0.5848572
Area
Rel. Res% (1): 1.116
4
400
300
2
3
200
1
100
0
Correlation: 0.99999
0
1000
Amount[ng/ul]
166
RRLC analysis of synthetic standards of synthetic C7-HSL
mAU
350
300
250
200
150
100
50
0
0.25
0.5
0.75
1
1.25
1.5
min
1.75
Retention time: 0.65 min
Report of calibration curve of C7-HSL
Compound #1,
Area = 0.48712438*Amt -71.822815
Area
Rel. Res% (1): 2.393
4
800
600
3
400
2
1
200
0
Correlation: 0.96827
0
1000
Amount[ng/ul]
167
RRLC analysis of synthetic standards of synthetic C8-HSL
mAU
35
30
25
20
15
10
5
0
0.5
1
1.5
2
min
2.5
Retention time: 1.75 min
Report calibration curve of C8-HSL
Com pound #1,
Area = 0.09296707*Am t +0.3828735
Area
Rel. Res% (1): 11.848
175
150
125
100
75
50
25
0
4
2
3
1
Correlation: 0.95965
0
1000
Amount[ng/ul]
168
RRLC analysis of synthetic standards of synthetic 3-oxo-C8-HSL
mAU
350
300
250
200
150
100
50
0
0.5
1
1.5
2
min
2.5
Retention time: 1.0 min
Report calibration curve of 3-oxo-C8-HSL
Compound #1,
Area = 0.83017507*Amt +3.9927246
Area
Rel. Res% (1): 9.5556e-1
4
1500
1250
1000
2
3
750
1
500
250
0
Correlation: 0.99999
0
1000
Amount[ng/ul]
169
RRLC analysis of synthetic standards of synthetic 3-hydroxy-C8-HSL
mAU
50
40
30
20
10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
min
Retention time: 0.85 min
Report calibration curve of 3-hydroxy-C8-HSL
Com pound #1,
Area = 0.09413402*Am t -0.0556122
Area
Rel. Res% (1): 8.1620e-2
175
150
125
100
75
50
25
0
4
2
3
1
Correlation: 1.00000
0
1000
Amount[ng/ul]
170
RRLC analysis of synthetic standards of synthetic C9-HSL
mAU
80
60
40
20
0
1
2
3
4
5
6
min
7
Retention time: 2.0 min
Report of calibration curve of C9-HSL
Com pound #1,
Area = 0.34098564*Am t +1.8802856
Area
Rel. Res% (1): 7.2748e-1
4
600
500
400
300
200
100
0
2
3
1
Correlation: 0.99998
0
1000
Amount[ng/ul]
171
RRLC analysis of synthetic standards of synthetic C10-HSL
mAU
100
80
60
40
20
0
1
2
3
4
5
6
min
7
Retention time: 3 min
Report calibration curve of C10-HSL
Com pound #1,
Area = 0.38160247*Am t +0.8328796
Area
Rel. Res% (1): 5.2429e-1
4
600
2
400
3
1
200
0
Correlation: 1.00000
0
1000
Amount[ng/ul]
172
RRLC analysis of synthetic standards of synthetic C11-HSL
mAU
90
80
70
60
50
40
30
20
10
0
0.2
0.4
0.6
0.8
1.2 min
1
Retention time: 0.8 min
Report of calibration curve of C11-HSL
Com pound #1,
Area = 0.14764265*Am t +1.8027252
Area
Rel. Res% (1): 2.260
4
250
200
2
150
100
3
1
50
0
Correlation: 0.99992
0
1000
Amount[ng/ul]
173
RRLC analysis of synthetic standards of synthetic C12-HSL
mAU
70
60
50
40
30
20
10
0
0.5
1
1.5
2
min
2.5
Retention time: 1.75 min
Report calibration curve of C12-HS:
Compound #1,
Area = 0.13821483*Amt +0.2098785
Area
Rel. Res% (1): 4.1611e-1
4
250
200
2
150
100
3
1
50
0
Correlation: 1.00000
0
1000
Amount[ng/ul]
174
RRLC analysis of synthetic standards of synthetic 3-oxo-C12-HSL
mAU
250
200
150
100
50
0
0.5
1
1.5
2
min
2.5
Retention time: 1.15 min
Report calibration curve of 3-oxo-C12-HSL
Com pound #1,
Area = 0.48609678*Am t -7.7326721
Area
Rel. Res% (1): -1.475
4
800
3
600
2
400
1
200
0
Correlation: 0.99982
0
1000
Amount[ng/ul]
175
RRLC analysis of synthetic standards of synthetic C13-HSL
mAU
100
80
60
40
20
0
0.5
1
1.5
2
min
2.5
Retention time: 0.7 min
Report calibration curve of C13-HSL
Com pound #1,
Area = 0.17506839*Am t +1.1098145
Area
350
300
250
200
150
100
50
0
Rel. Res% (1): 1.245
4
2
3
1
Correlation: 0.99998
0
1000
Amount[ng/ul]
176
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