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Microbial diversity and gene mining in Antarctic Dry
Valley mineral soils
Jacques J Smith
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Biotechnology,
University of the Western Cape
Bellville
Supervisor: Prof. D.A. Cowan
February 2006
i
ACKNOWLEDGEMENTS
My heavenly Father for His guiding hand and infinite wisdom.
My family, for their love, support and understanding through all the years of study
For Daleen, whose steadfast patience and support carried me through the trying times
Professor Don Cowan for having given me the opportunity and necessary guidance
The National Research Foundation and Ryoichi Sasakawa for providing the necessary
funding
Everyone in ARCAM, for humour and assistance and a memorable experience
ii
ABSTRACT
Microbial diversity and gene mining in Antarctic Dry Valley mineral soils
JACQUES J SMITH
PhD thesis, Department Biotechnology,
University of the Western Cape
Soil communities are regarded as among the most complex and diverse assemblages
of microorganisms with estimated bacterial numbers in the order of 109 cells.g-1.
Studies on extreme soils however, have reported lower cell densities, supporting the
perception that the so-called extreme environments exhibit low species diversity. To
assess the extent of microbial diversity within an extreme environment, the mineral
soils of the Dry Valleys, Ross Dependency, Eastern Antarctica were investigated using
16S rDNA analysis. Three mineral soils designated MVG, PENP and BIS were analysed,
each differing with respect to altitude, protein, lipid, water and DNA content. The midaltitude sample, MVG, yielded the highest levels of DNA and the low altitude BIS soil
contained the highest levels of protein, lipid and water. 16S clone libraries were
constructed and 60 unique clones were identified and sequenced. BLASTn analysis
revealed
eight
phylogenetic
groups
with
Cyanobacteria,
Actinobacteria
and
Acidobacteria representing the majority. The Cyanobacterial phylotypes were unique
to the desiccated high-altitude soils of the PENP sample, suggesting a soil-borne
Cyanobacterial population. 21% of the phylotypes identified were assigned as
‘uncultured’.
DNA isolated from the Antarctic mineral soils was also used to construct a
metagenomic clone library consisting of 90700 clones with an average insert size of
iii
3.5 kb, representing an estimated 3.4% of the available metagenome. Activity-based
screening of the library for genes conferring lipolytic activity yielded no positive
clones. It is suggested that the failure to produce positive clones might be a result of
insufficient nucleotide coverage of the metagenomic DNA.
The metagenomic DNA extracted from the Dry Valley mineral soils was further
analyzed using PCR. Two sets of degenerate primers based on conserved regions
within lipolytic genes were used to target lipase and esterase genes. One set of
primers was selected from a previous study. A second primer set was designed
manually from amino acid alignments of true lipase genes from family I, sub-families
I-VI. PCR analysis resulted in nine partial gene fragments varying between 240 bp and
300 bp. Bioinformatic analysis revealed that all nine partial gene fragments harboured
α/β-hydrolase motifs, putatively identifying two esterases and three lipases from both
bacterial and fungal origin.
iv
DECLARATION
I declare that Microbial diversity and gene mining in Antarctic Dry Valley mineral soils
is my own work, that it has not been submitted for any degree or examination in any
other university, and that all the sources I have used or quoted have been indicated
and acknowledged by complete references.
Jacques J Smith
24 February 2006
--------------------------------------
v
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
ii
ABSTRACT
iii
DECLARATION
v
LIST OF ABBREVIATIONS
x
LIST OF FIGURES
xiii
LIST OF TABLES
xiv
CHAPTER ONE:
LITERATURE REVIEW
1.1
Introduction
1
1.2
Antarctica
4
1.2.1
Antarctic Dry Valleys
5
1.3
Culture-independent analysis of environmental (metagemomic) source 7
1.3.1
16S rDNA sequencing
7
1.3.2
DNA denaturing and reassociation kinetics
8
1.3.3
G+C analysis
11
1.4
Metagenomic gene discovery
12
1.4.1
Enrichment
13
1.4.1.1
Sample enrichment
13
1.4.1.2
Genome enrichment
14
1.4.1.3
Gene targeting
16
1.4.2
Total community DNA extraction
17
1.4.3
Metagenomic DNA libraries
18
1.5
Psychrophiles
20
1.5.1
Molecular aspects associated with cold adaptation
22
1.5.1.1
Lipid composition
22
1.5.1.2
Protein stability
23
1.5.2
Biotechnological applications of psychrophiles
25
1.6
Lipases
27
1.6.1
Classification and taxonomy
27
1.6.2
True lipases
28
1.6.3
The GDSL family
29
1.6.4
Hormone sensitive lipase (HSL) family
30
1.6.5
Regulation of lipase expression
31
vi
1.6.6
Mechanism of secretion
32
1.6.6.1
Type I secretion
32
1.6.6.2
Type II secretion
33
1.6.6.2.1 Secretion across the inner membrane
33
1.6.6.2.2 Secretion across the outer membrane
33
1.6.6.3
The autotransporter pathway
34
1.6.7
Periplasmic folding
34
1.6.8
Psychrotrophic lipases
36
1.7
Biotechnological applications of lipases and esterases
38
1.7.1
Lipases in detergents
38
1.7.2
Biodiesel
39
1.7.3
Food industry
39
1.8
Aims of this study
41
CHAPTER TWO:
MATERIALS AND METHODS
2.1
Reagents
42
2.1.1
Chemicals
42
2.1.2
Antarctic soil samples (collection and storage)
42
2.1.3
Antibiotics
42
2.1.4
Enzymes
44
2.2
Culture Media
44
2.2.1
Luria-Bertani (LB) broth
44
2.2.2
LB agar
44
2.2.3
Terrific Broth (TB)
44
2.2.4
GYT Medium
45
2.2.5
Lipase specific medium
45
2.2.6
Esterase specific medium
45
2.3
Metagenomic DNA isolation
45
2.3.1
Modified Zhou method
45
2.3.2
Miller protocol
46
2.3.3
Bead beating protocol
47
2.4
Soil analysis
47
2.4.1
Dry weight assessment and water content
47
2.4.2
Protein assessment
48
vii
2.4.3
Lipid analysis
48
2.5
DNA quantification
48
2.6
PCR amplification using 16S rDNA primers
49
2.7
Agarose gel electrophoresis
49
2.8
GFX™ DNA purification
50
2.9
pMOSBlue blunt ended cloning
50
2.10
Preparation of electrocompetent E. coli cells
50
2.11
Preparation of chemically competent E. coli cells
51
2.12
Transformation of E. coli cells by:
52
2.12.1 Electroporation
52
2.12.2 Chemical
52
2.13
Colony PCR
53
2.14
Restriction endonuclease digestion
54
2.15
ARDRA analysis of 16S rDNA amplicons
54
2.16
Plasmid DNA extraction
54
2.16.1 Alkaline lysis
54
2.16.2 Talent kit
55
2.17
Nucleic acid sequencing
56
2.18
Preparation of metagenomic libraries
56
2.18.1 Metagenomic DNA digestion
56
2.18.2 A-tailing of 3’ termini of restriction digested metagenomic DNA
56
2.18.3 Recovery of restriction digested metagenomic DNA from agarose gel
57
2.18.4 5’-dephosphorilation of metagenomic DNA
57
2.18.5 Metagenomic library construction
57
2.19
Amplification of metagenomic library
58
2.20
Activity-based screening of the metagenomic library
58
2.21
PCR-based screening of the metagenomic library
59
2.22
PCR amplification of partial lipase fragments
59
CHAPTER THREE:
BACTERIAL DIVERSITY IN ANTARCTIC DRY VALLEY
MINERAL SOILS
3.1
Introduction
61
3.2
Aims
63
3.3
Results
63
viii
3.3.1
Soil properties
63
3.3.2
Cloning of the 16S rDNA PCR amplicons into pMOSBlue
65
3.3.3
16S rDNA ARDRA analysis
65
3.3.4
16S rDNA analysis and distribution
68
3.4
Discussion
69
3.4.1
Phylotype coverage
69
3.4.2
Phylotype distribution
71
CHAPTER FOUR:
METAGENOMIC LIBRARY CONSTRUCTION
4.1
Introduction
82
4.2
Aims
84
4.3
Results
84
4.3.1
Partial digestion and cloning of metagenomic DNA
84
4.3.2
Sequence space coverage
87
4.3.3
Library screening
88
4.3.3.1 Activity-based screening
88
4.3.3.2 PCR-based screening of the metagenomic library
88
4.4
88
Discussion
CHAPTER FIVE:
PROSPECTING
FOR
GENES
CONFERRING
LIPOLYTIC
ACTIVITY IN ANTARCTIC MINERAL SOILS
5.1
Introduction
94
5.2
Aims
95
5.3
Results
95
5.3.1
Degenerate primer design and PCR
95
5.3.1.1 Degenerate PCR using primer set OXF1 and ACR1
95
5.3.1.2 Design and PCR using primer sets LipF and LipR and LipF and LiR2
97
5.4
Analysis of partial lipase sequences
101
5.5
Discussion
104
CHAPTER SIX:
SUMMARY AND CONCLUSION
109
CONGRESS CONTRIBUTIONS
116
PUBLICATIONS
117
REFERENCES
118
ix
LIST OF ABBREVIATIONS
ABC
ATP-binding casette
ATP
adenosine triphosphate
BAC
Bacterial artificial chromosome
bp
base pair
BrdU
5-bromo-2-deoxyuridine
BSA
bovine serum albumin
CaCl2
calcium chloride
cm
centimeter
CP
cloud point
CTAB
cetyl-trimethyl-ammonium bromide
Da
Dalton
d
days
dNTPs
deoxyribonucleic-5'-triphosphate
DNA
deoxyribonucleic acid
DMSO
dimethyl sulfoxide
dNTP
deoxynucleoside triphosphate
DTT
ditriothritol
°C
degrees Celsius
EtOH
ethanol
EDTA
ethylene diamine tetraacetic acid
Fig.
figure
g
gram
GSP
general secretory pathway
GYT
glycerol yeast extract and tryptone
×g
centrifugal force
μg
microgram
H2SO4
Sulfuric acid
h
hour
HSL
Homoserine lactone
IPTG
isopropyl β-D-thiogalactosidase
kb
kilo basepairs
KCl
potassium chloride
kDa
kilo Dalton
x
kV
kilo volt
λ
lambda
l
liter
LB
Luria-Bertani
Lif
lipase specific foldase
µl
micro liter
M
molar
MCS
multiple cloning site
μF
micro Farad
ml
milliliter
min
minute
MgCl2
magnesium chloride
MgSO4
Magnesium sulfate
mM
millimolar
MnCl2
manganese chloride
m.s-1
meters per second
NaCl
sodium chloride
NaH2PO4
Sodium dihydrogen orthophosphate
NaI
sodium iodide
NaOH
sodium hydroxide
ng
nano gram
ng.μl-1
nano gram per micro liter
NH4AOc
Ammonium acetate
(NH4)2SO4 Ammonium sulphate
nt
nucleotide
Ω
Ohm
OD
optical density
PCR
polymerase chain reaction
PEG
poly ethylene glycol
pmol
picomole
rpm
revolutions per minute
RBS
ribosome-binding site
rDNA
ribosomal deoxyribonucleic acid
s
second
SAP
Shrimp alkaline phosphatase
xi
SDS
sodium dodecyl sulphate
SIP
Stable isotope probing
TA
Tris acetic acid
TAE
Tris acetic acid EDTA
TAIL
Thermal Asymmetric Interlaced PCR
TB
Terrific Broth
TBE
Tris boric acid EDTA
TCA
trichloroacetic acid
Tm
DNA dissociation temperature
TSS
transformation and storage solution
U
unit
UHQ
ultra high quality
UV
ultra violet
V
volts
VBNC
Viable but non-culturable
v/v
volume per volume
w/v
weight per volume
X-gal
5-bromo-4-chloro-3-indolyl-β-D-galactoside
y
year
xii
LIST OF FIGURES
CHAPTER 1
1.1
E.coli 16S ribosomal gene sequence and variable regions
9
1.2
Catalytic action of lipases
28
CHAPTER 3
3.1
Metagenomic DNA isolated from Antarctic Dry Valley mineral soils
66
3.2
PCR amplification of 16S rDNA genes from metagenomic DNA
66
3.3
Colony PCR analysis of recombinant pMOSBlue plasmids harbouring
16S rDNA genes
67
3.4
ARDRA analysis of 16S rDNA genes
67
3.5
Collectors curve of Antarctic Dry Valley mineral soil phylotypes
68
3.6
Pie chart representing Antarctic Dry Valley mineral soil phylotype
distribution
77
CHAPTER 4
4.1
Efficacy of metagenomic DNA extraction using alternate protocols
4.2
Metagenomic DNA smear generated following AluI restriction
endonuclease digestion
4.3
4.4
85
86
EcoRI Restriction endonulcease digestion of recombinant
PCR®-XL-TOPO® clones
87
PCR analysis of recombinant PCR®-XL-TOPO® clones
89
CHAPTER 5
5.1
Restriction endonuclease digestion of recombinant pGEM® – T easy
plasmids
96
5.2
Amino acid alignment of lipases used in degenerate primer design
98
5.3
PCR amplification using primers OXF1 and ACR1
100
5.4
PCR amplification using primers LipF and LipR2
100
5.5
Restriction endonuclease digestion of recombinant pTZ57T/A
101
xiii
LIST OF TABLES
CHAPTER 1
1.1
Growth temperatures of psychrophiles and psychrotrophs
21
1.2
Commercially relevant enzymes from psychrophiles
26
1.3
Cold-active lipases isolated from microorganisms
36
CHAPTER 2
2.1
Relevant plasmids, primers and strains used in this study
43
CHAPTER 3
3.1
Characteristics of the Dry Valley mineral soils
64
3.2
Unique 16S rDNA clone sequences identified in Antarctic mineral soils
72
3.3
Table of diversity indices
76
CHAPTER 5
5.1
Conserved motifs of true lipases from family I, subfamily I-VI
5.2
Bioinformatic analysis of the partial lipase gene fragments obtained
5.3
99
during PCR analysis
102
Nucleotide and protein sequences of the partial lipolytic gene fragments
103
xiv
Chapter 1
Literature Review
1.1 Introduction
It is widely acknowledged that soil microbial diversity in the environment (also referred
to as the metagenome) is severely underestimated. Initial studies to isolate and identify
the bacterial diversity in soils involved culturing without realizing the bias introduced by
selective culturing conditions. Identifying the bacteria present in various soil communities
would enable ecologists to cluster the microorganisms into operational taxonomic units
thereby defining which types are present (composition), number of types (richness) and
the frequency or relative abundance of each type (structure).
The paradigm of culture-based identification of bacteria in a given environmental sample
was first shifted fifteen years ago. Using reassociation kinetics on microbial community
DNA, Torsvik and co-workers (1990) estimated the bacterial diversity at 103 different
genomes per sample, a figure 200 times larger than that obtained for culturing alone. It
was concluded that standard plating techniques only accessed a minute fraction of the
soil bacterium flora. The inadequacy of soil community analysis by culturing was further
demonstrated by Amann et al. (1995) where they determined that microscopic
visualization of an environmental sample yielded cell counts two orders of magnitude
higher compared to culturing. Recently reanalysis of reassociation kinetics data of an
environmental sample indicated that the diversity was underestimated a thousand fold
assuming a lognormal distribution (Gans et al., 2005).
1
On the basis of rRNA gene sequence comparison, Woese (1987) showed that bacteria
could be classified into 12 divisions based on familiar culturable organisms such as
cyanobacteria, spirochetes and Gram-positive bacteria. During the past decade, these
divisions have expanded to 40, primarily due to culture-independent phylogenetic
surveys of microbial communities (Hugenholtz et al 1998). The limitations associated
with standard plate isolation strategies is that the specific culturing conditions required to
successfully isolate the majority of bacteria do not exist (Janssen et al., 2002).
Comparing the data from culturable and non-culturable bacteria, four cosmopolitan
divisions make up 90% of those bacteria readily cultured from soils (Proteobacteria,
Cytophagales, Actinobacteria and low G+C Gram positive bacteria). Other cosmopolitan
divisions such as Actinobacteria, Verrucomicrobia, GNS bacteria and OP11 are either
absent or poorly represented, even though division OP11 constitutes a major bacterial
group (Hugenholtz et al., 1998).
Resolving the total number of prokaryotic species on Earth is hindered by problems
regarding the definition of species as well as practical limitations associated with counting
prokaryotic species (Konstantinidis and Tiedje, 2004). Most of Earth’s prokaryotes are
found in the open ocean and in soil, where the numbers of cells are believed to be in the
order of 1029 to 1030, with the total number of prokaryotic cells on Earth estimated at 6 ×
1030 (Whitman et al., 1998). Problems in identifying and cataloguing all the prokaryotic
species stems from the fact that the prokaryotic species concept is not comparable to the
eukaryotic system (Konstantinidis and Tiedje, 2004). Of the 22 species concepts
established for eukaryotes, none is applicable to prokaryotes because prokaryotes lack
diagnostic morphological characteristics, proliferate asexually and exchange genetic
2
information in ways unusual to that of higher organisms (Brenner et al., 2000;
Stackebrandt et al., 2002). In addition, the metabolic and physiological properties of
prokaryotes are too poorly understood to accurately define phenotypic characteristics
required for species description (Vandamme et al., 1996). Additional limitations include
limited sampling of environments followed by attempts to extrapolate the data to a
global scale especially when the level of endemicity is still undetermined. High degrees of
endemicity would greatly expand Earth’s diversity. For example, a high level of
endemism has been recorded in the deep sea (Bull and Stach, 2004). Additionally, the
description of species based on ssu rRNA analysis is constraining mostly due to the 16S
rDNA molecule being too conserved to resolve species (Stackebrandt and Goebel, 1994).
Another obstacle facing culture-dependent analysis is the viable but not culturable
(VBNC) state of some bacteria. The VBNC state is believed to be either a process of selfpreservation whereby the bacterium ensures survival during environmental stress, or an
end-of-life-cycle process (McDougald et al., 1998). The argument is that if the former is
true, then resuscitation of the bacteria in the dormant VBNC state should occur when
conditions become favourable. Resuscitation has, however, not been conclusively proven
because it is unclear whether these ostensibly ‘uncultured’ cells have permanently lost
culturability (are dead), are killed by or are unable to grow on standard isolation media
or, are in a dormant state from which they might be recovered only if the correct
methodology is in place (Kell et al., 2004; Barer et al., 1998). Nevertheless, the
presence of bacteria in an environmental sample and the inability to culture all of them,
adds to the bias of culture-dependent studies (McDougald et al., 1998).
3
This chapter aims to highlight and discuss the current knowledge pertaining to the
biodiversity and environmental factors of the Antarctic Dry Valley mineral soils located in
the Ross Dependency, Eastern Antarctica. The review discusses the ongoing technologies
employed to assess global biodiversity within diverse environments, which include
culture-based analysis and constraints, 16S rDNA analysis, and reassociation kinetics.
The discussion concludes with approaches to exploit biodiversity within communities
using metagenomic gene discovery tools such as metagenomic enrichment strategies,
gene targeting and metagenomic libraries.
As a major section of this study centers on the diversity of cold active lipolytic enzymes
(EC 3.1.1.3), the final section of this review discusses the general characteristics of
lipolytic enzymes followed by the specific adaptations of these cold-active enzymes and
their biotechnological applications.
1.2 Antarctica
Antarctica is considered to be the last pristine continent. It has a land area of
approximately 4 × 106 km2 but expands its surface area 5.5 times to 22 × 106 km2 during
the austral winter due to ice formation (Friedmann, 1993). Of the 12.38 million km2 of
rock, 11.97 million km2 is covered in an ice sheet 2 km thick giving it the highest mean
elevation (2.3 km) of any continent. Extreme temperature fluctuations varying between
-50°C to 20°C and very high gravity-driven winds, referred to as katabatic winds, low
atmospheric humidity and low water potential are some of the factors which render
Antarctica one of the least hospitable environments on earth.
4
1.2.1 Antarctic Dry Valleys
Less than 2% of the Antarctic continent is permanently ice free (Cowan and Ah Tow,
2004). The largest ice-free area is the McMurdo Dry Valleys, situated in the Eastern
region of Antarctica (Doran et al., 2002). Other ice-free regions are found on the
Antarctic peninsula, Vestfold Hills, Bunger Hills, some coastal fringes and various
locations in the Transantarctic mountains. The Dry Valleys, collectively called the Ross
Desert, are the coldest and driest deserts on Earth and are subjected to harsh
environmental conditions (Wilson, 1970). The mean annual air temperature is -20°C,
with temperatures fluctuating around 0°C during summer months and dropping as low as
-55°C during winter. Ground surface temperatures during periods of direct sunlight
average 15°C but the air temperature seldom rises above 0°C (Wynn-Williams, 1990).
The upper mineral soil surface has a low water content (0.5%-2% wt) due to the
desiccating atmosphere. During winter months the high katabatic winds reduce humidity
to <10% which result in long periods of desiccation (Horowitz et al., 1972). Although
precipitation averages 15 g cm-1 year-1 in the form of snow, very little moisture reaches
the valley floor because of the high sublimation rate and low humidity. In addition, the
mineral soils are burdened by mineral salts such as sodium, calcium, magnesium,
chloride, sulfate and nitrate. The accumulation of these salts is due to upward
translocation from the substratum by capillary action and seaspray blown inland by
onshore winds (Vishniac, 1993).
The Dry Valley mineral soils are very nutrient poor as there is no plant life to liberate
organic matter. The presence of organic material would facilitate water retention and aid
to curb the high sublimation rate (Smith and Tearle, 1985). The aridity and low water
5
content of the Ross desert soils suggest low microbial abundance, as water is the limiting
factor in bacterial growth (Horowitz et al., 1972). Although temperate mineral soils
contain between 5% and at most 15% organic carbon, the Ross Desert soils contain
significantly less (≤0.05%) (Cameron, 1970). Sources of organic carbon in the Ross
Deserts are presumed to be exogenous as very few chemoautotrophs have been
documented. Cyanobacteria and algae have also been isolated, but photosynthetic
productivity appears to be confined to cryptoendolithic habitats in close proximity to
temporary or permanent water (Friedmann, 1993). For this reason it is presumed that
organic matter influx in the Ross Desert is airborne-driven from aquatic habitats. The
aerial dispersal of cyanobacterial mats from ice-covered lakes in Taylor Valley has been
estimated at 2.93 × 104 kg.y-1 (Kappen and Straka, 1988).
Antarctic soils are largely aerobic and the occurrence of anaerobic bacteria is considered
rare (Line 1988). Early culture-based analysis by Baker and Smith (1972) indicated that
seventy-one percent of the bacteria present in Antarctic soil were related to coryneforms
within the genera Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium and
Kurthia. Some Bacillus and Micrococcus species as well as Pseudomonas, Flavobacterium
and other Gram-negative aerobic rods such as Alcaligenes and Agrobacterium were also
identified (Baker and Smith, 1972). In the Ross Desert the highest numbers of culturable
isolates are present in the permafrost layer and towards the surface. Chromogenic
bacteria are most prevalent at the soil surface whereas non-pigmented bacteria are
mainly located below the soil surface (Cameron et al., 1972). Bacteria isolated from the
McMurdo
Dry
Valleys
most
commonly
belong
to
the
genera:
Achromobacter,
Arthrobacter, Bacillus, Corynebacterium, Flavobacterium, Micrococcus, Planococcus,
6
Pseudomonas, Streptomyces, and Nocardia of which Coryneforms represent the most
abundant bacteria present (Cowan and Ah Tow, 2004).
1.3
Culture
independent
analysis
of
environmental
(metagenomic)
sources
1.3.1 16S rDNA sequencing
Initial attempts at determining microbial diversity relied on the direct extraction and
sequencing of 5S rRNA molecules from environmental samples (Stahl et al., 1985).
However, the mere 120 bp of the 5S rRNA molecule provided limited information due to
the paucity of independently varying nucleotides, limiting its usefulness to simple
ecosystems. Subsequently, an approach using the 16S rRNA gene for phylotype
identification was suggested (Pace at al., 1986; Olsen et al., 1986). The 16S rRNA gene
has an average length of 1500 nucleotides, yields substantially more information than
the 5S rRNA gene, and contains highly conserved sequence domains interspersed with
nine variable regions (Fig 1.1) (Van de Peer et al., 1996). This allowed for more detailed
analysis and identification of signature sequences over a wide taxanomic range.
Advantages in using rRNA genes for analysis include: 1) They occur in high copy
numbers (from 1 to 10 per cell), 2) sequence data can be obtained without prior
cultivation and 3) 16S rDNA sequences have been determined for a large number of
bacteria (Amann et al., 1995).
The most widely adopted method for obtaining 16S rDNA sequences is through the use of
PCR. Total community DNA is isolated from the environment and the 16S rRNA genes
amplified using universal primers designed to target the conserved regions within the
7
rRNA genes (Fig 1.1). The PCR amplicons are cloned and recombinant clones are
sequenced and compared to one other and sequences within the 16S rDNA database
(Altschul et al., 1997). Although PCR analysis of community DNA bypasses culturing and
is a rapid process for generating vast amounts of data, several factors have been
identified which can skew phylogenetic analysis. Rainey et al. (1994) suggested that high
percentage G+C DNA templates are discriminated against due to low efficiency of strand
separation during denaturation. Target sequences that are in higher abundance are
preferentially amplified and cloning sticky-end products may produce better results than
blunt-end products (Rainey et al., 1994). Amplification of rRNA genes using standard PCR
conditions have been found to exclude other important environmental taxa due to
primers designed to target only known bacteria (Reysenbach et al., 1992) and Suzuki
and Giovannoni (1996) indicated that the amount and origin of background DNA in a
sample could affect results by out competing the 16S rDNA target sequences. The
formation of chimeras has also been reported (Maidak et al., 2000). Although, computer
programs have been developed to eliminate chimeric sequences, chimeras with greater
than 85% homology are not easily identified (Theron and Cloete, 2000).
1.3.2 DNA denaturation and reassociation kinetics
DNA denaturation and reassociation kinetics have been used to determine the
heterogeneity of a given environmental sample and so provide a broad-scale analysis of
community structure (Torsvik et al., 1990; Øvreås et al., 2003). It does not, however,
provide information relating to the phylogenetic origin of the microorganisms present. As
the isolated community DNA is denatured, the optical density of the DNA is recorded over
time and plotted against temperature (usually between 65°C and 95°C) and
8
KEY:
totally conserved
conserved
variable
> 75% variable
priming sites
10
20
30
variable
highly
variable regions
40
50
60
70
AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGT
100
110
120
130
140
E9F
AACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGAT
80
150
160
170
180
190
200
210
V1
GGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTC
260
270
280
V2
GGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGAC
220
230
290
300
240
250
310
320
330
340
350
GATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAC
360
370
380
390
400
410
420
GCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCT
430
440
450
460
470
480
490
TCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCC
550
560
V3 540
GCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAA
500
510
520
530
570
580
590
600
610
620
630
TTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAA
V4
CTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
640
650
710
720
660
670
730
740
680
690
750
760
700
770
TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGC
780
790
800
810
820
830
840
GTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCC
850
870
880
890
900
910
940
950
960
970
980
V5
CTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAAC
920
930
TCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACC
990
1000
1010
1020
1030
1040
1050
TTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTG
1100
1110
1120
V6 1090
CTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCC
1060
1070
1080
1130
1140
1150
1160
1170
1180
1190
TTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATG
1200
V7
1210
1220
1230
9
1240
1250
1260
ACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCG
1270
1280
1290
1300
1310
1320
1330
ACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCAT
1350
1360
1370
1380
1390
1400
V8
GAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACC
1340
1410
1420
1430
1440
1450
1460
1470
GCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACT
1540
V9 1530
TTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCT
1480
1490
1510
1520
U1510R
TA
Fig 1.1.
1500
Escherichia coli 16S rRNA gene sequence annotated with “universal” bacterial
priming sites (E9F and U1510R). Variable regions V1-V9 are colour coded to
indicate bacterial sequence variability (Van de Peer et al., 1996).
displayed as a sigmoidal curve. Using the information gathered upon DNA denaturation
the first derivative of the melting curve is calculated and the melting profiles converted
to mol %G+C as described by Mandel et al. (1970). The first derivative of the melting
curve provides information concerning the %GC distribution within the community. A less
complex sample for instance, might display a single melting point which results in a
steep slope on the sigmoidal curve. A more complex community containing a variety of
genomes will have different melting points and produce a shallower slope (Øvreås et al.,
2003). Genomic DNA of known mol %G+C is used as standard in both DNA denaturation
and reassociation studies.
Following denaturation, the DNA is allowed to reassociate. The principle of reassociation
kinetics is based on the rate of DNA hybridization within the sample, where the rate of
hybridization is proportional to the concentration of complementary DNA sequences and
inversely proportional to the length of different sequences (Theron and Cloete, 2000).
DNA reassociation is measured over time and the fraction of reassociated DNA (C/C0) is
expressed as a function of C0t, where C0 is the initial molar concentration of single-
10
stranded DNA nucleotides. The reaction rate constant, k, can be expressed as 1/C0t1/2,
where t1/2 denotes the time in seconds required for 50% reassociation. The genomic DNA
standards are then used to calculate the size of the community genome relative to that
of the standard (Britten et al., 1974)
1.3.3 G+C analysis
When only a coarse level of resolution of a community is required, G+C analysis is
typically used. The technique is based on the fact that prokaryotic genomic DNA varies
between 24% and 76% with respect to G+C vs. A+T and that particular taxonomic
groups do not vary more that 5% in G+C content (Vandamme et al., 1996). G+C
analysis is considered to be a coarse analysis because several taxonomic groups may
have a similar G+C range. G+C range does, however, correlate with certain physiological
attributes. Microorganisms with a high G+C range (60-75%) are generally obligate
aerobes with an oxidative metabolism, while organisms with a fermentative metabolism
largely have low G+C content (Santo Domingo et al., 1998). The method is based on the
principle that bis-benzimidazole binds to adenine and thymidine thereby altering the
buoyant density of the DNA molecule in proportion to its A+T content (Holben and Harris,
1995). A gradient of DNA fragments of different G+C concentrations is subsequently
established using equilibrium density-gradient (CsCl) centrifugation. The G+C content of
each fraction is established by using a standard curve relating G+C content to density.
G+C analysis can be applied to all the extracted DNA in the environmental sample and it
is not subject to the biases of PCR-based methods. The method is quantitative and
provides a means for detecting sparse members of a community since it separates low
biomass fractions from dominant fractions (Tiedje et al., 1999).
11
1.4 Metagenomic gene discovery
Strategies to find and isolate novel enzymes from the environment involve enrichment,
isolation and screening of a wide variety of organisms in pure culture for the desired
activity. To increase the probability of obtaining the desired gene a range of culturing and
enrichment conditions need to be set up. This includes incubation at various
temperatures, levels of pH, and supplementing media with different carbon sources
under both aerobic and anaerobic conditions (Wilkinson, 2002). Upon identification of the
microorganism possessing the desired activity, the gene is then isolated. Although this
method is a common route to discovering new enzymes a substantial fraction of the gene
diversity could be lost due to inefficient culturing conditions and VNBC organisms
(McDougald et al., 1998; Janssen et al., 2002).
To overcome culturing constraints, several DNA-based molecular methods have been
developed, primarily focusing on 16S RNA genes. Although 16S RNA analysis provides
information regarding the species present, little to no information regarding the
functionality or phylogenetic relationship of the bacteria can be determined (Streit and
Schmitz, 2004).
Metagenomic gene discovery circumvents culture-based enzyme screening techniques by
targeting DNA directly in the environment as outlined below. The generation of large
insert libraries allows cloning of large (40–150 kb) DNA fragments from environmental
samples, theoretically isolating genes from any origin, sequence and function (Streit and
Schmitz, 2004). Direct genomic cloning not only provide access to biotechnologically
useful genes, but also to operons or genes encoding pathways that direct the synthesis
12
of complex molecules such as antibiotics. Furthermore, sequence space flanking a
particular gene of interest can easily be obtained and/or the phylogenetic origin of the
functional gene can be determined (Streit and Schmitz, 2004). Examples of metagenomic
gene discovery include the cloning of DNA from uncultured soil microorganisms and the
identification of five clones conferring 4-hydroxybutyrate dehydrogenase (Henne et al.,
1999) and three clones conferring lipolytic activity (Henne et al., 2000). Rondon et al.
(2000) used a BAC vector to isolate genes conferring DNase, lipase and amylase activity
from soil. Eleven clones exhibiting lipolytic activity towards tributyrin were identified
following the construction of a metagenomic library from pond water (Ranjan et al.,
2005) and Majerník and co-workers (2001) identified a single gene conferring Na+/H+
antiporter activity by screening metagenomic libraries using a Na+/H+ antiporter deficient
E. coli host.
1.4.1 Enrichment
1.4.1.1 Sample enrichment
Sandaa and co-workers (1999) determined, using reassociation kinetics, that heavy
metal contaminated soils contained approximately 2000 genomes.g-1 soil whereas noncontaminated soils could contain as much as 16 000 genomes.g-1. Thus, a gene of
interest represents a very small fraction of the nucleic acid pool due to the high degree of
genetic diversity present in soils. This diminishes the likelihood of obtaining that gene
upon expression screening of metagenomic libraries. Pre-enrichment of the sample
enhances the probability of detecting a desired gene. One such strategy is culture
enrichment, which is aimed at the culturable fraction of microorganisms in an
environment. Selection pressure is based on nutritional, physical or chemical criteria.
13
Two separate studies used selective enrichment strategies to isolate genes conferring
alcohol oxidoreductase (Knietsch et al., 2003) or dehydratase activity (Knietsch et al.,
2003). Using indicator agar-plate assays, these investigators identified sixteen different
clones utilizing 1,2-propanediol and two clones possessing the glycerol or diol
dehydratase genes, respectively.
Other forms of enrichment include specific whole-cell enrichment where, for example,
size selective filtration was used during the Saragasso Sea genome sequencing project to
omit eukaryotic cells (Venter et al., 2004). Similarly, enrichment for Buchnera aphidicola
was achieved by differential centrifugation, followed by whole genome sequencing
(Schloss and Handelsman, 2003).
1.4.1.2 Genome enrichment
Genomic enrichment is also aimed at the active components of a microbial community.
During a process called stable-isotope probing (SIP) the genomic DNA or RNA of specific
members of a community is selectively labeled. (Boschker and Middelburg, 2002).
Isotopes of
13
C,
18
O and
15
N are used to label substrates such as phenol, methanol,
ammonia and methane. The isotopes are incorporated in the DNA and RNA of the actively
growing microorganisms and the labeled nucleic acids are separated using density
gradient centrifugation. SIP was first used to identify
13
C-enriched phospholipid fatty
acids signature profiles in experiments aiming to identify microbial populations
responsible for acetate oxidation in sediments (Boschker et al., 1998). A previously
unknown group of phenol degraders, Thauera, was identified using
13
C-phenol in an
anaerobic bioreactor by enriching populations with RNA-SIP (Manefield et al., 2002). Pelz
14
et al. (2001) also employed SIP to identify sulfate-reducing bacteria in hydrocarboncontaminated aquifers using
13
C-toluene as substrate. SIP substrates are expensive and
limited in availability and alternatives such as 5-bromo-2-deoxyuridine (BrdU) can be
used. BrdU is a structural analogue of thymidine and the uptake of [3H]thymidine has
routinely been used for measuring the in situ growth of bacteria in various environments
(Smalla, 2004). BrdU incubation was used to detect metabolically active bacteria in lake
water (Borneman, 1999). BrdU labelled DNA or RNA can also be separated using density
gradient centrifugation or immunocapture. Although these methods prove effective, loss
of specific enrichment may occur due to cross feeding and recycling of the label within
the community (Smalla, 2004).
Another approach, suppressive subtractive hybridisation (SSH), identifies genetic
differences between microorganisms. SSH can be used to successfully identify specific
genes in closely related bacteria such as genes responsible for pathogenesis or xenobiotic
degradation (Cowan et al., 2005). Two separate restriction digested fractions are
generated termed ‘driver’ and ‘tester’, each ligated to unique adaptors. The ‘driver’ is
present in excess (usually 100 fold) and is used to trap and eliminate the background in
the ‘tester’ by associating with identical DNA sequences during hybridisation. Unique
heteroduplex DNA association between ‘driver’ and ‘tester’ represents the DNA sequences
of interest (Felske, 2002). Galbraith et al. (2004) demonstrated that SSH could also be
used to identify differences between complex DNA samples.
15
1.4.1.3 Gene targeting
Targeting genes with specific metabolic or biodegradative capabilities using PCR has been
extensively used. Sheu et al. (2000) detected polyhydroxyalkanoate synthases from
nineteen PHA-positive strains by designing degenerate primers. Gene-specific PCR can
also be used in environmental samples enabling the detection of specific genes. Bell et al.
(2002) designed degenerate primers using conserved amino acid regions within lipase
genes. Using these degenerate primers they obtaining partial lipase gene fragments from
a metagenomic soil sample, and obtained the full-length lipase genes using genomewalking PCR (Morris et al., 1995). Although proven to work well (Hallin and Lindgren
1999; Henckel et al., 2000), this approach is limiting with respect to biocatalyst
discovery. Firstly, primer design is limited to available sequences. Functionally similar
genes from distant families or genes resulting from convergent evolution are unlikely to
be detected using a single gene-family-specific set of primers. Very distant gene families
tend to require separate family-specific primers as primers with excessive degeneracy
(>256 fold) seldom yield results. Gene-specific PCR only yields a partial gene fragment,
requiring additional steps to obtain the up- and down-stream flanking regions. In such
instances, the partial gene fragment can be used as a probe to identify possible fulllength genes in a metagenomic library or enzyme restricted metagenomic DNA, which
can be excised and cloned. More direct approaches for obtaining up- or down-stream
sequences are PCR-based and include strategies such as genome walking PCR (Kilstrup
and Kristiansen, 2000), Thermal Asymmetric Interlaced PCR (TAIL) (Liu and Whittier,
1995), panhandle PCR (Megonigal et al., 2000) and inverse PCR (Hartl and Ochman
1994).
16
1.4.2 Total community DNA extraction
The first step toward gene targeting is nucleic acid extraction. Although numerous nucleic
acid extraction methods exist, extraction is achieved using two types: cell extraction and
direct lysis (Roose-Amsaleg et al., 2001). During cell extraction, whole cells are
separated from the sample prior to lysis and DNA retrieval. It is a time consuming
process and generally includes laborious methods such as differential centrifugation and
sucrose gradients to obtain whole cells (Steffan and Atlas, 1988). Direct lysis methods do
not require cell separation. The DNA yield is much higher and more representative of the
microbial community compared to the yield obtained using cell extraction (Steffan et al.,
1988). This is largely attributed to the fact that a greater number of the microorganisms
are subjected to lysis, notably those embedded in soil particles (Roose-Amsaleg et al.,
2001). In addition, non-bacterial DNA, such as from fungal or plant material is also
isolated. A major drawback of direct lysis is that other organic components such as humic
and fulvic acids are co-extracted. These contaminants inhibit downstream reactions,
especially PCR, and require additional purification such as gel extraction or PVPP
treatment (Steffan et al., 1988). The mainstream methods of choice in direct lysis are
the Miller (Miller et al., 1999) and Zhou (Stach et al., 2001) protocols. The Miller protocol
is a mechanical process which involves bead beating and tends to access a higher degree
of diversity because Gram-positive and other bacteria such as Micromonospora sp. are
insensitive to chemical lysis treatment (Niemi et al., 2001). However, chemical lysis has
shown to be more efficient in extracting high molecular weight DNA (Stach et al., 2001).
Total DNA extracted directly from environmental samples does not contain equal
distribution of the communities’ genomes, which generally leads to bias in downstream
applications such as PCR (Suzuki and Giovannoni, 1996).
17
1.4.3 Metagenomic DNA libraries
The same principles for single genome DNA library construction (generation of DNA
fragments via shearing or enzyme digestion, cloning of DNA fragments into a suitable
vector and screening for genes of interest) apply for metagenomic library construction.
The first study in which metagenomic cloning was reported involved the cloning of a 40
kb genome fragment from a planktonic marine archaeon to characterize uncultivated
prokaryotes (Stein et al., 1996). However, the intended downstream application(s)
dictates the vector and host systems used. Protocols that yield highly fragmented DNA
(0.5-5.0
kb)
severely
limits
applications
of
the
isolated
DNA,
especially
when
downstream processing involves restriction digestion to generate sticky ends, which will
result in significant loss of the total DNA complement. To accommodate randomly
fragmented DNA, cloning systems designed for blunt-end or T/A ligation could be used.
Where restriction digestion is a requisite, high molecular DNA is paramount, for example
partial digestion using Sau3AI and subsequent ligation to BamHI prepared vector. The
latter approach was successfully applied in screening and detecting single gene products
permitting between 104 and 106 clones to be screened (Henne et al., 1999). Other
cloning systems such as cosmids, fosmids and bacterial artificial chromosomes (BACs)
are widely used in library construction and accommodate large inserts (25-45 kb for
cosmids and fosmids, >40 kb for BACs). Large inserts enable capture of more sequence
space and allow for detection of whole metabolic pathways, such as the biosynthetic
gene cluster for the synthesis of the antibiotic violacin (Brady et al., 2001).
Construction and screening of single or metagenomic DNA libraries typically relies on E.
coli as expression host. This host is favoured because it is the best characterized in terms
18
of biochemistry and genetics, having served as a biological model for cellular processes
such as DNA replication, transcription, metabolic pathways and signal transduction
(Thompson and Zhou, 2004). However, the limitations associated with E. coli as an
expression host are underlined by the low number of positive clones obtained during a
single round of screening, usually less that 0.01% (Cowan et al., 2005). The best chance
for recovering heterologously expressed genes in E. coli is from native promoters or
read-through transcription from vector based promoters. The probability, however, of
expressing a heterologous gene due to a translational fusion is extremely low and would
statistically involve the screening of >107 clones (Gabor et al., 2004). This suggests that
the discovery of target genes from a complex metagenomic sample without sample
enrichment is technically challenging.
E. coli transcriptional machinery is known to be relatively promiscuous in recognizing
foreign expression signals, but with a strong bias towards Firmicutes genes (Gabor et al.,
2004). Other important factors for efficient heterologous gene expression include the
efficiency with which the –35 and –10 DNA elements of the promoter correlate with the
E. coli consensus sequences TTGACA and TATAAT, respectively (Gross et al., 1992). If
heterologous expression relies on transcription from a vector-encoded promoter, the
foreign gene must be inserted in the correct orientation with respect to the promoter.
Conversely, if expression is dependent on a native promoter it must be recognized by the
host RNA polymerase holoenzyme (Old and Primrose, 1994). Factors governing
translation such as initiation codons and ribosome-binding site (RBS) are also important
in successful expression. However, given that the characteristics of the genes cloned
during metagenomic library construction are unknown, translational factors could
19
contribute to lack of expression. The introduction and expression of foreign DNA in a host
organism often change the metabolism of the organism in ways that may impair normal
cellular function due to increased metabolic load. This typically occurs upon over
expression of the foreign DNA due to high copy number of the plasmid or transcription
from a strong plasmid-based promoter (Glick, 1995). Such a high level of expression of a
foreign protein can initiate a cellular stress response, including increased synthesis of
cellular proteases, so that the recombinant protein is rapidly degraded (Glick, 1995).
Careful choice of expression plasmid and promoter would circumvent such a problem and
enhance the probability of obtaining positive clones.
1.5 Psychrophiles
Psychrophilic organisms are widely distributed and inhabit both terrestrial and aquatic
environments. Such habitats exist in polar and alpine regions, much of the worlds
oceans, subterranean systems such as caves, the upper atmosphere, and man-made
systems (Cavicchioli et al., 2002). Due to huge debate surrounding the distinction
between psychrophilic and psychrotolerant, one of the earliest criteria used to define
psychrophilic microorganisms is the ability to form visible colonies on solid media within
1 to 2 weeks at 0°C. Although this is no longer a definitive characteristic it illustrates that
psychrophiles are defined by their minimum growth temperature and not by their
optimum or maximum growth temperature as with mesophiles and thermophiles.
Currently, the definition of psychrophile by Morita (1975) is widely accepted (Helmke and
Weyland, 2004) (Table 1.1). Determining the minimum growth temperatures of
psychrophiles and psychrotrophs is difficult because of their very slow growth rates.
Nutrient availability is also important as the media composition can alter the minimum
20
growth temperature. The lowest temperature of bacterial growth is thought to be -12°C
(Russell, 1992) for at lower temperatures the intracellular ice formation in the cytoplasm
and an increase in solute concentrations would prevent growth.
Table 1.1
Minimum,
optimal
and
maximum
growth
temperatures
of
psychrophilic
and
psychrotrophic microorganisms
Minimum
Optimum
Maximum
Psychrophilic
<0°C
<15°C
<20°C
Psychrotrophic
3-5°C
>15°C
>20°C
The ability to grow at low temperatures is widespread amongst microbial flora, which
include representatives from the eubacteria, yeasts, fungi, algae and Archaea. Like the
ability to perform photosynthesis, it seems probable that the ability to grow at low
temperatures has evolved independently in several microorganisms (Russell, 1992).
Additionally, species within a single genus are known to be psychrophilic, mesophilic or
thermophilic, for example the genera Clostridium and Bacillus amongst the eubacteria
and the cyanobacterial genus Phormidium (Russell, 1992).
Psychrophilic and psychrotolerant microorganisms are not only widespread in the
environment. Many well-known food-spoilage and food-poisoning bacteria are known to
thrive in cold environments. Food-spoilage bacteria include Brochothrix thermosphacta
and P. fragi. B. thermosphacta is favoured in packed meat and meat products due to
their tolerance to low pH, curing salts and chill-storage. Listeriosis is caused by Listeria
monocytogenes, which grow at temperature around 10°C, particularly in cold raw milk,
21
soft cheese and processed poultry (Glass and Doyle, 1989). Yersinia enterocolitica is
capable of growth and toxin production at temperatures as low as 4°C and causes
yersiniosis,
which
manifests
as
gastroenteritis
in
adults
and
peritonitis,
ileitis,
pseudoappendicitis in children following the ingestion of contaminated milk (Wannet et
al., 2001).
1.5.1 Molecular aspects associated with cold adaptation
1.5.1.1 Lipid composition
Temperature has a major influence on both the fluidity and phase behaviour of
membrane lipids. As microorganisms cannot insulate themselves, they are required to
regulate their lipid composition to maintain the activity of their membrane-associated
proteins. The primary thermal responses of membrane lipids are alterations in fatty acyl
composition, which in the event of a growth temperature drop result in increased
unsaturation, reduction in average chain length, increase in methyl branching and an
increase in the ratio of antesio branching relative to iso branching (Russell and
Hamamoto, 1998). The most common response to a decrease in temperature is the
accumulation of unsaturated fatty acids in membrane lipids (Nedwell, 1999). The level of
unsaturation is controlled by cold-shock fatty acid desaturases thereby regulating the
homeostasis of the membrane fluidity. Desaturases are encoded by four genes, desA-D,
are membrane bound in bacteria and algae and target intact acyl lipids as substrates
(Foot et al., 1983; Los, 2004). The introduction of double bonds into fatty acids by
desaturase action allows for rapid response upon a decrease in membrane fluidity
allowing adaptation to a sudden drop in temperature. Cell membranes are essentially
anisotropic solutions of phospholipids and proteins in a fluid phase and it is only within
22
the fluid phase that they are biologically functional. As temperature decreases,
membranes become increasingly viscous which leads to a decrease in membrane fluidity
(Sinensky, 1974) and at a certain temperature, depending on the physiology of the
microorganism, the membrane phase will shift to solid and biological function lost. The
alterations in membrane lipids thus allow maintenance of biological function, a feature
called homeoviscous adaptation (Sinensky, 1974).
1.5.1.2 Protein stability
The stability and flexibility required for enzymes to adapt to various temperature ranges
are largely controlled by amino acid substitutions. Although past research mainly focused
on mesophilic and thermophilic enzymes, studies on psychrophilic enzymes revealed that
the same general changes in amino acid composition apply. Obtaining the flexibility
required for catalysis in cold environments is achieved by reversing the stabilizing factors
required for rigidity in thermophilic enzymes: These include fewer ionic interactions and
intramolecular hydrogen bonds (Cavicchioli et al., 2002). To aid in destabilization coldactive enzymes contain more polar and/or charged side groups, which promote
interaction with solvents (Fields, 2001). Additional strategies to enhance entropy in
psychrophilic enzymes involve a reduction in proline residues while increasing the
number of glycines (Haney et al., 1999b). The increase in flexibility and lowering of
stability in cold-active enzymes results in increased activity at low temperatures and low
thermostability at elevated temperatures (Gerike et al., 2001). Although the alterations
listed above are used by enzymes to adapt to all ranges of temperature, one potential
region for change, the active site, however, remains conserved across broad taxonomic
groups and all temperature ranges. It appears that substrate binding is so specific that
23
any changes might lead to significant reduction or outright loss in catalytic activity,
inferring that the altered or substituted residues must occur some distance from the
active site.
Homology modeling of Arthrobacter species’ citrate synthase, a well studied psychrophilic
enzyme, exhibits an additional surface loop compared to its mesophilic counterpart from
M. smegmatis. The loop consists of nine amino acids of which six are charged. On
another surface loop, present in both enzymes, the Arthrobacter enzyme has nine
substitutions of neutral with charged amino acids to further enhance surface interaction
(Gerike et al., 2001). Except for the latter changes, no additional charged amino acids
are present in the psychrophilic enzyme. Other expected changes in thermostability such
as increased number of isoleucine residues or changes in the arginine:lysine ratio were
not observed. Although the proline distribution differed between the two enzymes,
nothing could be deduced based on homology modeling. Crystal structure analysis
revealed that the overall structures of the two enzymes were similar. The active site of
the psychrophilic enzyme had however, a larger opening due to shortening of a loop on
one side and the substitution of an arginine with an alanine. As was expected, the
structure and integrity of the active sites were retained. However, the surface
surrounding the entrance was more negatively charged, aiding in forcing substrates into
the pocket. Overall, the surface of the cold-active enzyme was more hydrophobic,
causing destabilization by means of entropy-driven ordering of the surrounding solvent
water molecules thus contributing to low temperature activity (Russell et al., 1998;
Russell, 2000; Gerike et al., 2001).
24
1.5.2 Biotechnological applications of psychrophiles
Table 1.2 lists some of the biotechnologically relevant enzymes from psychrophilic
prokaryotes. Only within the past few years has it been recognized that psychrophilic
microorganisms and their enzymes provide a reservoir for biotechnological exploitation
(Gounot, 1991; Margesin and Schinner, 1994). Possible applications include:
K Enzymes such as lipases, cellulases and proteinases used in detergents
K Enzymes as flavour-modifying agents
K Biosensors for environmental applications
K Environmental bioremediation
K Food processing, such as cheese manufacture and meat tenderizing
Advantages of using psychrophiles and/or their enzymes in biotechnological applications
include:
K Heat lability of psychrophilic enzymes allows for rapid and economic termination of
processes using moderate heat treatment
K Less expensive processing due to elimination of expensive heating and/or cooling
processes
25
Table 1.2
Examples of commercially relevant enzymes from psychrophiles. Data obtained
from Cavicchioli et al. (2002), Gerike et al. (2001) and Bhat (2000)
Enzyme
Organism
Application
Triose phosphate isomerase
Vibrio marinus
Biotransformation
Lipase
Aspergillus nidulans
Food, detergents, cosmetics
α-Amylase
Alteromonas haloplanktus
Pectinase
Sclerotinia borealis
Alkaline phosphatase
Vibrio sp. I5
Pulp bleaching, starch
hydrolysis
Cheese ripening, wine
industry
Molecular biology
Cellulase
Fibrobacter succinogenes
Animal feed, textiles
Xylanase
Cryptococcus adeliae
Wine industry, fermentation
Nitrile hydatase
Rhodococcus sp. N-774
DNA ligase
Pseudoalteromonas
haloplanktis
Low-temperature acrylamide
synthesis
Molecular biology
Although a wide range of potential applications and benefits can be gained from coldactive enzymes, very few of the benefits have been exploited. This is primarily due to a
lack of understanding of the cellular behavior and molecular structure of psychrophiles
compared with thermophilic counterparts (Russell and Hamamoto, 1998).
One aspect of psychrophilic proteins and their biotechnological potential which have been
studied is that of ice-nucleation. Ice-nucleating proteins produced by P. fluorescens, X.
campenstris and Erwinia herbicola have substantial biotechnological potential. These
plant pathogens induce frost damage in crops by triggering ice formation on leaf and
flower surfaces (Li and Lee, 1995). Ice nucleating proteins are encoded by ina genes,
although the mechanism of ice formation remains unclear (Wolber and Warren, 1989;
Kajava and Lindow, 1993). Possible biotechnological applications for ice-nucleating
26
proteins include ice-cream manufacture, synthetic snow, freeze-texturing, freeze-drying
and concentrating (Russell and Hamamoto, 1998). Using natural or genetically
engineered bacteria deficient in ice formation as frost protectants for sensitive plants to
out-compete natural ice-nucleating pathovars is another application which has shown
potential (Gurian-Sherman and Lindow, 1993).
1.6 Lipases
1.6.1 Classification and taxonomy
Until recently, enzymes were classified as true lipases (EC 3.1.1.3) based on two criteria.
Firstly, activity increased in the presence of lipid:water interfaces, a phenomenon termed
‘interfacial activation’ (Brzozowski et al., 1991) and secondly, lipases exhibit a ‘lid’
domain, which is a surface loop covering the active site which was displaced upon
contact with the interface (Derewenda et al., 1994). These criteria became untenable due
to a number of exceptions where enzymes having a ‘lid’ did not exhibit interfacial
activation
(Verger,
1997).
For
this
reason,
lipases
are
generally
defined
as
carboxylesterases capable of catalyzing both the hydrolysis and synthesis of long-chain
acylglycerols (Fig. 1.2). Glycerol esters with an acyl chain of ≥10 carbon atoms are
regarded as lipase substrates and those of ≤10 carbon atoms as esterase substrates, with
trioleolglycerol being the standard lipase substrate under laboratory conditions (Jaeger et
al., 1999). The presence of esterases is normally indicated by the hydrolysis of
glycerolesters such as tributyrylglycerol (tributyrin) (Jensen, 1983). It should be noted,
however, that most lipases are also capable of hydrolyzing esterase substrates.
27
Fig. 1.2 The catalytic action of lipases. The hydrolysis of a triglyceride results in glycerol and fatty
acids. The reverse synthesis reaction is also possible by combining fatty acids with
glycerol to form a triglyceride. Taken from Jaeger and Reetz, 1998.
1.6.2 True lipases
Lipolytic enzymes form part of the α/β-hydrolase superfamily (Ollis et al., 1992) and are
distinguished by a catalytic triad Ser-Asp-His. The serine residue usually occurs in a
conserved pentapeptide Gly-Xaa-Ser-Xaa-Gly. Lipolytic enzymes are classified into 8
families broadly based on size, function and manner of secretion. Family I is further
categorized into six subfamilies of which subfamilies 1, 2 and 3 constitute true lipases,
and subfamilies 4, 5 and 6 lipases and phospholipases from Gram-positive bacteria. P.
aeruginosa is the prototypical lipase of subfamily I.1 and has a molecular mass ranging
between 30-32 kDa. Other lipases belonging to subfamily I.1 that display high amino
acid similarity to P. aeruginosa are produced by V. cholerae, A. calcoaceticus, P.
wisconsinensis and P. vulgaris. Subfamily I.2 lipases have slightly higher molecular
weights (33 kDa) owing to an amino acid insertion forming an anti-parallel double βstrand at the surface of the molecule (Nobel et al., 1993). Lipases of both family I.1 and
I.2 require the co-expression of a chaperone protein termed lipase-specific foldase (Lif)
to correctly fold into an active state (Rosenau et al., 2004). Exceptions are lipases from
28
P. luteola, P. vulgaris, P. fragi and P. fluorescens C9, for which no Lif has been identified.
Another distinguishing feature of subfamilies I.1 and I.2 is the two aspartic residues
involved in a Ca2+-binding site are conserved. Two cysteine residues, which form a
disulphide bridge, are conserved in the majority of lipase sequences. The residues
involved in Ca2+-binding and disulphide bridge formation are located near the catalytic
His and Asp residues and are believed to be important in active site stabilization (Kim et
al., 1997). Secretion of subfamilies I.1 and I.2 lipases is mediated through the general
secretory pathway (Gsp). Subfamily I.3 contains enzymes from two species, P.
fluorescens and S. marcescens, with molecular weights of 50 kDa and 65 kDa,
respectively. Not only are they larger than those of subfamilies I.1 and I.2, but they do
not contain cysteine residues and have no N-terminal signal peptide for proteolytic
cleavage during type II secretion. Due to the lack of an N-terminal signal peptide,
secretion is mediated through the ATP-binding-cassette (ABC) transporter system
(Duong et al., 1994).
1.6.3 The GDSL family
GDSL lipases of family II do not exhibit the signature pentapeptide Gly-Xaa-Ser-Xaa-Gly.
These enzymes contain a Gly-Asp-Ser-(Leu) [GDS(L)] motif, incorporating the active site
serine residue. The serine residue in these enzymes is situated much closer to the Nterminus than in other lipolytic enzymes (Upton and Buckley, 1995). The esterase from
S. scabies is also included in this family due the 30% similarity it shows towards the
Aeromonas hydrophila esterase. Crystal structures of the S. scabies GDSL esterase show
a catalytic dyad instead of a triad in the catalytic center (Wei et al., 1995). Both the
enzymes from S. scabies and A. hydrophila have an α/β tertiary fold substantially
29
different from that of the α/β-hydrolase family (Arpigny and Jaeger, 1999). The latter
two enzymes share conserved sequence blocks with three other bacterial esterases from
P. aeruginosa, S. typhimurium and P. luminescens. Secretion of these lipases are
mediated through the autotransporter system where the C-terminal of the protein
mediates secretion by the formation of 12 amphipathic β-sheets to form a pore in the
outer membrane.
1.6.4 Hormone sensitive lipase (HSL) family
Lipases belonging to families IV-VI are all classified as HSL lipases. The most striking
characteristic of family IV lipases is their high amino acid similarity to mammalian HSL
(Hemilä et al., 1994). Family V enzymes originate from across the temperature spectrum
and include lipases form psychrophilic (Moraxella sp., Psychrobacter immobilis),
mesophilic (Pseudomonas oleovorans, H. influenzae, A. pasteurianus) and thermophilic
(S. acidocaldarius) bacteria.
Family V share significant amino acid similarity (20-25%) to various bacterial nonlypolytic enzymes such as epoxide hydrolases and dehalogenases, which also possess the
typical α/β-hydrolase fold and a catalytic triad (Misawa et al., 1998). Family VI enzymes
represent some of the smallest esterases known and range from 23-26 kDa. The 3D
structure of P. fluorescens carboxylesterase indicated that the active form is a dimmer
(Kim et al., 1997) and hydrolyses small substrates exhibiting broad specificity. Limited
studies have been performed on the other enzymes in this family as their amino acid
sequences were derived from whole-genome sequences. Family VII esterases are large
(55 kDa) and share significant amino acid sequence homology (30% identity) to
eukaryotic acetylcholine esterases and intestine/liver carboxylesterases. The esterase
30
from Arthrobacter oxydans is active against phenylcarbamate herbicides and is plasmid
borne enabling possible transfer to other species or strains (Pohlenz et al., 1992). Family
VIII is represented by three enzymes which show striking similarity to class C βlactamases. A 150 amino acid stretch is 45% similar to an Enterobacter cloacae ampC
gene suggesting that the esterases in this family possess an active site more reminiscent
of that found in class C β-lactamases, which involve a Ser-Xaa-Xaa-Lys motif (Galleni et
al., 1988).
1.6.5 Regulation of lipase gene expression
The regulation of lipase gene expression has been studied in S. aureus, Streptomyces
species and A. calcoaceticus. Lipase production is thought to be mediated by quorum
sensing and two-component transduction. Quorum sensing is a cell density response
system associated with a cascade of other genes, primarily genes associated with
virulence (Weingart et al., 1999). Many Gram-negative bacteria produce extracellular
signaling molecules called autoinducers which belong to the class of acylated homoserine
lactones (AHL’s). AHL’s bind to regulator proteins which induce or repress certain specific
target genes. The best-studied example of lipase gene regulation is the lipase operon of
P. aeruginosa. Extracellular lipase is detected when P. aeruginosa enters stationary
phase, which is suggestive of cell density-dependant regulation (Stuer et al., 1986).
Gene fusion studies with LipA::LacZ revealed that lipase gene expression was controlled
by the quorum sensing activator RhlR and the autoinducer N-butyryl-homoserine lactone
(BHL). RhlR activated the transcription of the two-component regulatory system LipR. In
turn, LipR regulated lipase transcription by binding to an upstream activating sequence
preceding the σ54-dependant promoter of the lipase operon (Jaeger et al., 1999;
31
Rosenau and Jaeger, 2000). The global regulator protein GacA has also been implicated
in lipase regulation where overproduction of GacA led to increased levels of extracellular
lipase (Reimmann et al., 1997).
1.6.6 Mechanism of secretion
All known bacterial lipases are extracellular enzymes. This requires translocation across
the single cytoplasmic membrane of Gram-positive bacteria or both the inner and outer
membranes, enclosing the periplasmic space of Gram-negative bacteria. Different
mechanisms of protein export have evolved to direct bacterial enzymes to their final
destination of which three (type I, II and V) are involved in lipase secretion. Efficient
secretion of enzymatically active enzymes is tightly coupled to correct folding and involve
specific and unspecific periplasmic folding mediated by enzyme-specific chaperones.
1.6.6.1 Type I secretion
Type I secretion pathways, also referred to as ATP-binding cassette (ABC) protein export
systems, are employed by a wide range of different Gram-negative bacteria and are
responsible for the secretion of toxins, proteases and lipases (Fath and Kolter, 1993;
Binet et al., 1997). The proteins secreted by the type I pathway are not subject to
proteolytic cleavage and therefore lack cleavable N-terminal leader peptide sequences
(Binet et al., 1997). Instead, the secretion signal is located within the C-terminal 60
amino acids of the secreted protein (reviewed by Binet et al., 1997; Duong et al., 1996).
32
1.6.6.2 Type II secretion
1.6.6.2.1 Secretion across the inner membrane
Type II secretion involves a two step process whereby proteins first cross the inner
membrane with the aid of Sec proteins (Pugsley, 1993), followed by translocation across
the outer membrane via either Xcp proteins (Filloux et al., 1998) or autotransporter
proteins (Henderson et al., 1998).
The Sec secretion system of E. coli has been extensively studied and consists of a
cytoplasmic secretion-specific chaperone (SecB), a protein translocation ATPase (SecA)
and an integral membrane protein complex formed by at least six different protein
subunits (SecY, SecE, SecD, SecF, SecG and YajC) (reviewed by Pugsley et al., 1997;).
1.6.6.2.2 Secretion across the outer membrane
Following export of proteins, which are to be secreted, into the periplasm via the Sec
translocase system, the proteins may undergo further modifications before they are
finally translocated across the outer membrane. This final translocation step requires
several accessory proteins, collectively referred to as the type II secretion apparatus or
secreton. This apparatus is highly specific and is capable of distinguishing proteins to be
secreted from resident periplasmic proteins and, with a few exceptions, it can
discriminate between its own secreted proteins and those induced from other species
(Lindeberg et al., 1996; Filloux et al., 1998).
33
The type II secretion systems are widely distributed and appear to be the primary
pathway for the secretion of extracellular degradative enzymes by Gram-negative
bacteria (Hobbs and Mattick, 1993; Pugsley et al., 1997; Russel, 1998).
1.6.6.3 The autotransporter pathway
Bacterial proteins that are targeted to the microbial surface or released into the
environment often depend on periplasmic proteins and almost always require outer
membrane proteins to promote their secretion. The autotransporter family of Gramnegative bacterial proteins is a unique subset of secreted proteins that do not rely on
other proteins for transit from the periplasm to the bacterial surface (reviewed by
Henderson et al., 1998; Jacob-Dubuisson et al., 2001). The esterase, EstA, produced by
P. aeruginosa is an enzyme secreted by the type V autotransporter pathway and contains
the characteristic GDSL active site consensus motif (Arpigny and Jaeger, 1999). EstA and
other type V proteins possess a C-terminal domain that mediates targeting to and
translocation across the outer membrane. All presently known autotransporter proteins
originate from pathogenic bacteria and encompass diverse functionalities and include
proteases, toxins, adhesins and invasins (Henderson et al., 1998).
1.6.7 Periplasmic folding
Lipases secreted via the secreton-mediated pathway fold into an active conformation in
the periplasm before they are translocated across the outer membrane. To achieve this
secretion-competent
conformation,
lipases
require
specific
intermolecular
folding
catalysts, Lif proteins. These foldases are encoded in the same operon as their cognate
lipases and have been identified in several bacteria. Lifs represent a unique family of
34
proteins with no significant homology towards other classes of proteins and are grouped
into four families (Rosenau et al., 2004). Algorithms used in protein production reveals
that all Lifs appear to possess a similar secondary structure, with 70% consisting of αhelical and 30% of random coil elements (Frenken et al., 1993). The N-terminus of Lifs
contain a predicted hydrophobic transmembrane section which is thought to anchor the
Lif to the inner membrane with almost the entire protein exposed to the periplasm.
However, membrane anchoring does not appear vital as Lifs with truncated N-terminal
domains or fused with signal sequences to allow translocation into the periplasm, were
still able to activate their cognate lipases when expressed in the host strain. The precise
role of lipase activation by Lifs is difficult to determine in vivo due to folding and
secretion being such a tightly coupled process. Current knowledge is based on expression
studies of various lipase/Lif systems in heterologous hosts as well as in vitro experiments
using purified lipases and Lifs. Findings demonstrated that Lifs and their cognate lipases
form stable complexes that can be co-purified or co-immunoprecipitated (El Khattabi et
al., 2000) and that Lifs mediate refolding of chemically denatured lipases in vitro (Ihara
et al., 1995). Lipases are not only dependent on these intermolecular chaperones, they
show
distinct
specificity,
since
the
chaperones
of
different
species
are
not
interchangeable (El Khattabi et al., 1999). To date, the mechanism whereby these
foldases recognize their cognate lipases is unknown, which prompted the theory that
disulfide bonds might play an important role in lipase/foldase recognition. This however
is not the case as cysteine-to-serine variants were able to adopt an active conformation,
implying that disulfide bonds are not necessary for the recognition of the lipase by its
foldase nor for the interaction of both proteins (Liebeton et al., 2001).
35
1.6.8 Psychrotrophic lipases
Table 1.3 lists the psychrotrophic lipases isolated over the past 15 years. Although
lipases are considered the third most important group of enzymes in biotechnology next
to proteases and carbohydrases (Hasan et al., 2005), there are currently only a few
documented psychrophilic lipases. The latest report on the isolation of a cold-active
lipase is that by Kulakova and co-workers (2004) where they isolated a Psychrobacter sp.
from Antarctic soil exhibiting lipolytic activity. Isolation and characterization of the
enzyme indicated the presence of a cold-active esterase. To date all studies involved in
the isolation of cold-active lipolytic enzymes relied on isolation of a pure culture
exhibiting lipolytic activity on selected media. Following identification of the isolates, they
proceeded to isolate and characterize the lipolytic genes by constructing single genome
Table 1.3
Cold-active lipases isolated from microorganisms over the past fifteen years
Origin
Microorganism
Enzyme
Reference
Antarctica
Moraxella TA144
Lipase
Feller et al., 1991
Antarctica
Psychrobacter immobilis B10
Lipase
Arpigny et al., 1993
Alaska
Pseudomonas sp. B11-1
Lipase
Choo et al., 1998
Soil isolate
Aspergillus nidulans WG312
Lipase
Mayordomo et al., 2000
Water isolate
Pseudomonas sp. Strain KB700A
Lipase
Rashid et al., 2001
Siberia
Acinetobacter sp. Strain No. 6
Esterase
Suzuki et al., 2002
Dairy products
Pseudomonas fragi
Lipase
Alquati et al., 2002
Antarctica
Psychrobacter Ant 300
Esterase
Kulakova et al., 2004
36
libraries and screening for expression in E. coli. This has led to the isolation of various
lipolyic enzymes of which lipases appear to be more frequent (Table 1.3). The cold-active
lipolytic enzymes isolated vary with respect to size and range between 32 kDa and 68
kDa and all display the characteristic Gly-Xaa-Ser-Xaa-Gly motif associated with
esterase\lipase proteins of the α/β-hydrolase superfamily (Arpigny and Jaeger, 1999).
The esterase from Psychrobacter Ant300 shows 44% identity to the Moraxella TA144
lipase and groups, together with Acinetobacter sp. Strain No. 6, with the HSL lipase
family. The optimum catalytic activity of all the enzymes varied with Moraxella TA144
lipase exhibiting the lowest optimum at 17°C and Acinetobacter sp. Strain No. 6 esterase
and Psychrobacter immobilis B10 lipase exhibited the highest at 45°C. The pH range of all
the cold-active enzymes isolated ranged from neutral to slightly alkaline (pH 6-9). All but
one of the enzymes, Pseudomonas sp. Strain KB700A lipase, showed activity towards
esters of short chain fatty acids (≤C6), whereas Strain KB700A indicated high activity
towards long chain fatty acids (C18). Structural analysis of the Pseudomonas fragi lipase
indicated the presence of 24 arginine residues, more than twice the number present in
the P. aeruginosa and B. cepacia mesophilic enzymes. 20 of the 24 arginine residues
were found to be evenly distributed across the surface of the protein, thus increasing the
amount of charged residues and enhancing flexibility in cold environments (Gerike et al.,
2001). Of the cold-active lipolytic enzymes isolated, the lipase from A. nidulans is the
only one used in industry and is used in food processing, detergents and the manufature
of cosmetics (Mayordomo et al., 2000).
37
1.7 Biotechnological applications of lipases and esterases
Microbial lipases and esterases constitute an important group of biotechnologically
valuable enzymes because of the versatility of their applied properties and ease of mass
production. Lipases are valued biocatalysts because they act under mild conditions, are
highly stable in organic solvents, have diverse substrate specificity and they display high
levels of stereo-, chemo- and regioselectivity in catalysis (Jaeger and Eggert, 2002;
Jaeger and Reetz, 1998).
1.7.1 Lipases in detergents
The most commercially important field of application for hydrolytic lipases is in
detergents for both household and industrial use. An estimated 1000 tons of lipases are
added to the approximately 13 billion ton of detergents annually (Godfrey and West,
1998). To improve the efficacy of detergents, modern heavy-duty detergents usually
contain one or more enzymes such as protease, amylase, cellulase and lipase (Jaeger
and Reetz, 1998). Using enzyme cocktails in detergents is environmentally friendly due
to a reduction in undesirable chemicals, biodegradability of the enzymes, a non-negative
impact on sewage treatment processes and the enzymes present no risk to aquatic life
(Hasan et al., 2005). Lipases active under alkaline conditions, such as the A. oryzae
derived lipase, are preferred because laundering conditions usually occur between pH 1011 (Satsuki et al., 1990). Other lipases used in detergents originate from Candida sp.
and Chromobacterium.
38
1.7.2 Biodiesel
Another potential application for lipases is the production of biological diesel. The process
entails the production of diesel from vegetable oils. Currently, interest in biodiesel is
mostly environmental as the fuel exhibits almost total absence of sulphur and does not
contribute to new carbon dioxide emissions (Nabi et al., 2006). The production of
biodiesel involves the conversion of vegetable oil into methyl- or other short-chain
alcohol esters by a single transesterification reaction catalyzed in an organic solvent
(Jaeger and Eggert, 2002). This is however, an energy consuming process also requiring
the addition of esters drawback mono and diglycerides, glycerol, water and an alkaline
catalyst (Salis et al., 2005). Another drawback is the low-temperature property of the
fuel. The triacylglycerol source for the production of the diesel plays an important role, as
esters from saturated fatty acids crystallize at higher temperatures than that of
unsaturated esters (Salis et al., 2005). The ratio of saturated to unsaturated fatty acids
determined the temperature at which the fuel starts to freeze or cloud point (CP) of
biodiesel. If the biodiesel mixture is inadequate the CP will be too high and additives such
as butyl oleate must be added to maintain fluidity (Linko et al., 1998). Although
immobilized P. cepacia lipase has shown promise in overcoming most of the drawbacks,
industrial scale production has not yet been attained due to the high cost of the
biocatalyst (Hasan et al., 2005).
1.7.3 Food industry
Lipases are used to improve the quality of a number of food products such as fruit juices,
baked foods, fermented vegetables and dairy products (Zalacain et al., 1995). The
position, chain length and degree of saturation greatly influence the nutritional and
39
sensory value of a given triglyceride (Jaeger and Reetz, 1998). The hydrolysis of milk fat
in dairy products results in flavour enhancement in certain cheeses, especially that of
soft cheese. The flavour is produced by the free fatty acids released during lipolysis of
the cheese. The addition of lipases which release short chain fatty acids (C4 and C6)
generate a sharp, tangy flavour, where as the release of medium chain fatty acids (C12
and C14) imparts a soapy taste (Hassan et al., 2005).
40
1.8 Aims of this study
The mineral soils of Antarctica have been extensively studied using culture-based
approaches (Friedmann, 1993). Molecular phylogenetic analysis has allowed for increased
access to microbial diversity in all biotopes including other Antarctic niches such as
cryptoendolithic (de la Torre et al., 2003), cryoconite holes (Christner et al., 2003) and
lake ice (Priscu et al., 1998) and lake (Brambilla et al., 2001) communities. Extreme
environments such as the Arctic potentially harbour a novel nucleic acid pool with
biotechnological potential. One such important biocatalyst is lipase of which several have
been isolated from similar environments (Feller et al., 1991; Choo et al., 1998; Kulakova
et al., 2004). All of the cold-active lipolytic enzymes isolated to date, however, have
been accessed using culture-based techniques. In an effort to better understand bacterial
community structure in Antarctic Dry Valley mineral soils, and probe the mineral soil
metagenome using PCR-based methodology the primary aims of this investigation were
the following:
(i)
Determine and compare the bacterial diversity of Antarctic Dry Valley mineral soil
samples.
(ii)
Screen the Antarctic Dry Valley mineral soils for the presence of genes conferring
lipolytic activity.
41
Chapter 2
Materials and Methods
2.1 Reagents
2.1.1 Chemicals
Table 2.1 lists all the strains, plasmids and primers used in this study. Unless otherwise
stated, all chemicals used were of analytical grade and obtained from MERCK laboratory
supplies. Ingredients used for microbial media was obtained from MERCK (biolab
diagnostics), Oxoid (Oxoid ltd) or KIMIX laboratory supplies.
2.1.2 Antarctic soil samples (collection and storage)
Antarctic mineral soil samples were collected from three dry mineral soil sites: (i)
underneath a Crab-eater seal carcass on Bratina Island (BIS1); (ii) the mid-slopes of
Miers Valley (MVG); and (iii) fine gravels from Penance Pass, a high altitude site between
the Miers and Shangri La Valleys (PENP) in sterile 500 ml Nalgene® tubes Samples were
recovered under aseptic conditions by removal of a 1 cm surface layer of mineral soil
from a 20 x 20cm sample area. All samples (approx. 400g) were mixed thoroughly and
resampled before storage at <0oC for transport. Samples were stored at -80oC until
required.
2.1.3 Antibiotics
Supplementation of antibiotics to either broth or growth media was performed
aseptically. The appropriate filter sterilized antibiotic was added to autoclaved broth or
42
media following cooling to ~ 45°. Antibiotic final concentrations were: ampicillin, 100
μg.ml-1; kanamycin, 50 μg.ml-1 and tetracycline, 15 μg.ml-1.
Table 2.1
Bacterial strains, plasmids, and primers used in this work.
Strains/Plasmids/Primers
Relevant Characteristics
Source/Reference
DH5α
recA endA1 hsdR17 supE4 gyrA96 relA1
Δ(lacZYA-argF)U169 (φ80dlacZΔM15)
Promega
pMOSBlue
endA1 hsdR17(rk12-mk12+)supE44 thi –1
recA1 gyrA96 relA1 lac[F’ proA+B+
lacIqZΔM15:Tn10(TcR)]
F- mcrA Δ(mrr-hsdRMS-mcrBC)
φ80lacZΔM15 ΔlacX74 recA1 deoR
araD139 Δ(ara-leu)7697 galU galK rpsL
(Strr) endA1 nupG
Amersham
Biosciences
Plasmids:
pMOSBlue
PCR-XL-TOPO
pTZ57R/T
lacZ’ ApR
lacZ’ ccdB KmR
lacZ’ ApR
Amersham
Invitrogen
Fermentas
Primers:
E9F
U1510R
5’ – GAGTTTGATCCTGGCTCAG – 3’
5’ – GGTTACCTTGTTACGACTT –3’
Farelly et al., 1995
Reysenbach and
Pace, 1995
Bell et al., 2001
Bell et al., 2001
This study
This study
This study
α
www.IDT.com
α
www.IDT.com
α
www.IDT.com
α
www.IDT.com
UCT
Strains:
E. coli
Top10
#
Invitrogen
Corporation
(Carlsbad, CA, USA)
OXF1
5’ – CCYGTKGTSYTNGTNCAYGG – 3’
ACR1
5’ – AGGCCNCCCAKNGARTGNSC – 3’
Δ
5’ – GACCRATYGTSCTSGTVCAYGG – 3’
LipF
*
LipR
5’ – GACATGRCCNCCYWKGCTRTC – 3’
*
LipR2
5’ – GCCRCCSTGRCTRTGRCC – 3’
M13F
5’ – GTTTTCCCAGTCACGAC – 3’
M13R
5’ – CAGGAAACAGCTATGAC – 3’
M13F sequencing
5’ – CGCCAGGGTTTTCCCAGTCACGAC – 3’
M13R sequencing
5’ – GAGCGGATAACAATTTCACACAGG – 3’
β
5’ – GCCAGCAGCCGCGGTAATAC – 3’
F3:16.5
#
Designed to target the conserved oxyanion hole region of the lipase gene
$
Designed to target the conserved active site region of the lipase gene
Δ
Modification of the OXF1 primer by lesstening the degeneracy and so narrow the specificity to
true lipase sequences
*
Modification of the ACR1 primer by lesstening the degeneracy and so narrow the specificity to
true lipase sequences
α
Manufactured by Integrated DNA Technologies in the USA
β
Sequencing primer designed at the University of Cape Town sequencing facility to bind internally
at bp 517-536 of the 16S rDNA gene (Figure 1.1)
$
43
2.1.4 Enzymes
The following enzymes were used in DNA manipulation: Restriction enzymes and T4 DNA
ligase (Fermentas), Shrimp Alkaline Phosphatase (SAP) (Roche). Thermostable BIO-XACT™ DNA polymerase and 10 × reaction buffer was obtained from Bioline.
2.2 Culture Media
2.2.1 Luria-Bertani (LB) broth
LB broth consisted of 1% (w/v) tryptone, 0.5% (w/v) yeast extract and 1% (w/v) NaCl.
All constituents were mixed together with ultra high quality (UHQ) Millipore water and
the pH adjusted to 7.0 using 10 N NaOH prior to autoclaving.
2.2.2 LB agar
LB agar consisted of LB broth prepared as above (Section 2.2.1) with the addition of
1.3% bacteriological agar prior to autoclaving.
2.2.3 Terrific Broth (TB)
TB was prepared according to Sambrook et al. (1999) and consisted of 1.2% (w/v)
tryptone, 2.4% (w/v) yeast extract, 0.4% glycerol (v/v), 17 mM KH2PO4 and 72 mM
K2HPO4 in UQH Millipore water. Tryptone, yeast extract and glycerol were made up
separately in 900 ml UHQ before autoclaving. Following cooling to room temperature all
solutions were mixed.
44
2.2.4 GYT medium
GYT medium was prepared according to Tung and Chow (1995) and contained 10% (v/v)
glycerol, 0.125% (w/v) yeast extract and 0.25% (w/v) tryptone. The GYT medium was
mixed thoroughly, filtered using a 0.22 μ filter and stored at 4°C until required.
2.2.5 Lipase specific medium
Lipase agar plates were prepared according to Kouker and Jaeger (1987) with slight
modification. The medium was prepared by mixing 1% (w/v) tryptone, 0.5% (w/v) yeast
extract, 0.5% NaCl, 1% (v/v) olive oil, 0.001% rhodamine B and 0.1% gum Arabic after
which the pH was adjusted to 7.0 and the medium autoclaved. Where required, the
appropriate antibiotic was added after cooling of the medium before it was poured into
plates. Plates were stored at 4°C until used.
2.2.6 Esterase specific medium
Agar plates for esterase screening contained the same constituents as lipase plates
except the olive oil and rhodamine B was replaced with 1% tributyrin (v/v).
2.3 Total community DNA Isolation
2.3.1 Modified Zhou method
Community DNA extractions were performed according to the modified Zhou protocol
(Stach et al., 2001). Aliquots of mineral soils (5 g) were weighed out into sterile 30 ml
Nalgene® centrifuge tubes followed by the addition of 6.75 ml soil extraction buffer (1%
CTAB [w/v]; 100 mM Tris, pH 8.00; 100 mM NaH2PO4, pH 8.00; 100 mM EDTA; 1.5 M
NaCl; 0.02% Protease K [w/v]). The tubes were incubated horizontally at 37°C for 30
45
min with shaking. 750 μl 20% [w/v] SDS was added to each tube followed by a further 2
h incubation at 65°C with gentle inversions every 20 min. Following incubation, the tubes
were centrifuged at 3000 × g for 10 min at room temperature and the supernatant
pooled
into
a
sterile
Nalgene®
30
ml
centrifuge
tube.
An
equal
volume
Phenol/Chloroform/Isoamyl was added and mixed gently followed by centrifugation at 16
000 × g for 10 min. Supernatants were again transferred to sterile Nalgene® 30 ml
centrifuge tubes with the addition of an equal volume of chloroform. After careful mixing
the tubes were centrifuged at 16 000 × g for 10 min at room temperature and
supernatants recovered. Chloroform washes were repeated until the supernatants were
clear. Once all washes were complete 0.6 volumes of isopropanol was added to the
supernatants
and
DNA
precipitation
allowed
to
take
place
overnight
at
room
temperature. DNA was pelleted by centrifugation at 10 000 × g for 10 min, washed with
70% ethanol, recentrifuged at 10 000 × g for 5 min, and air dried in a sterile hood. UHQ
Millipore water was used to resuspend the DNA pellet and a small fraction was analysed
by gel electrophoresis.
2.3.2 Miller protocol
DNA extraction from all three soils were extracted using the Miller protocol (Miller et al.,
1999). Between 0.5 and 1 g of soil was added to sterile 2 ml screw cap tubes containing
0.5 g sterile Quartz sand, followed by 300 μl phosphate buffer, pH 8.00, 300 μl lysis
solution (0.5 M Tris-HCl, pH 8.00, 10% SDS [v/v], 100 mM NaCl) and 300 μl chloroform.
The sample tubes were mixed and either shaken in a bead beater (Bio101 FastPrep
FP120, Savant Instruments Inc. Holbrook, NY) at 4.5 m.s-1 for 40 s or vortexed for 1 –
1.5 min at full speed, followed by centrifugation for 5 min at 13 000 × g. Supernatants
46
were transferred to clean 1.5 ml Eppendorf tubes with the addition of 7 M NH4AOc to
achieve a final concentration of 2 M. Tubes were inverted several times until white
flocculates appeared and centrifuged for 5 min at 13 000 × g. The supernatants were
recovered and transferred to clean centrifuge tubes after which 0.6 volumes of
isopropanol was added, the tubes inverted several times and incubated at room
temperature for 15 min. DNA was collected at room temperature by centrifugation at 13
000 × g for 10 min, washed with 70% EtOH and air dried in a sterile hood. UHQ Millipore
water was used to resuspend the air-dried DNA pellet and a small fraction was analysed
by gel electrophoresis.
2.3.3 Bead beating protocol
Environmental DNA was extracted from mineral soils using the FastDNA spin kit for soil
(Bio101 Inc., Vista, CA, USA) and bead beater (Bio101 FastPrep FP120, Savant
Instruments Inc. Holbrook, NY). Extraction was performed as described in the
manufacturer’s instructions for soil DNA extraction and DNA eluted in UHQ Millipore
water.
2.4 Soil analysis
2.4.1 Dry weight assessment and water content
Dry weights were determined by placing 10 g samples of mineral soil sample, in
duplicate, in pre-weighed glass petri dishes. The samples were incubated at 100°C and
weighed every 24 h for a period of 3 days using a Mettler-Toledo PE 360 balance. Water
content was determined the total difference in soil sample weight, expressed as a
percentage.
47
2.4.2 Protein assessment
Total protein of each of the soil samples was determined using the Bio-Rad protein assay
kit (Bio-Rad). 200 μl of the supernatant of DNA extractions was used in the microassay
procedure as outlined by the manufacturer. Optical density measurements were
performed at 595 nm and plotted against a 1-20 μg BSA standard curve.
2.4.3 Lipid analysis
Total lipid was extracted according to Folch et al. (1957). 20 ml of chloroform:methanol
(2:1 [v/v)]) was prepared and added to 0.5 g of each soil sample. Following 15 min
shaking at room temperature the samples were centrifuged at 3000 × g for 5 min and the
supernatant removed. The solvent phase was then washed with 0.2 volumes 0.9%
aqueous NaCl and centrifuged at 1000 × g for 5 min. The upper phase was aspirated and
the interphase washed twice with methanol:water (1:1). Following the final wash,
centrifugation and removal of the upper phase, the lower chloroform phase containing
extracted lipids was allowed to evaporate overnight in the fume hood. Lipid content was
determined gravimetrically.
2.5 DNA quantification
Extracted, dried community DNA was resuspended in sterile UHQ H2O and allowed to
stand overnight. Uncut commercial λ-DNA of known concentration was used as a DNA
standard to determine the quantity of total community DNA extracted. λ-DNA was diluted
to a concentration of 5 ng.μl-1 and loaded at 5, 10 and 15 ng aliquots into a 1% (w/v)
agarose gel. Dilutions of 1:10 and 1:100 of community DNA was loaded adjacent to the
48
λ-DNA standards and analyzed following agarose gel electrophoresis for 4 min at 80 V.
For more sensitive DNA analysis, quantification was performed using the Nanodrop ND1000. The instrument was blanked using 1 μl of the same UHQ Millipore water as for DNA
resuspension or elution. 1 μl of resuspended or eluted DNA was then added to the
reading platform and the DNA concentration recorded.
2.6 PCR amplification using 16S rDNA primers
High molecular weight community DNA was used a template for 16S rDNA amplification
using primers E9F (Farely et al., 1995) and U1510R (Reysenbach and Pace, 1995). PCR
reactions (25 μl) contained ~ 10 ng template DNA, 1 × NEB PCR buffer (20 mM Tris-HCl
(pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1% [v/v] Triton X – 100), 0.16
mM of each dNTP, 160 ng BSA, 0.5 pmol of each primer and 0.5 μl Taq DNA polymerase.
PCR reaction mixtures were placed in an Applied Biosystems thermocycler Gene Amp®
2700 using the following PCR cycling conditions: Initial denaturation at 95°C for 90 s,
followed by 28 cycles of denaturation at 94°C for 20 s, annealing at 50°C for 40 s, and
extension at 72°C for 1 min. The final elongation step was performed at 72°C for 3 min. A
positive reaction containing E. coli genomic DNA as template DNA was included. For
control purposes, a reaction mixture containing all reagents except template was
routinely included. An aliquot of each reaction mixture was analysed using gel
electrophoresis as described in Section 2.7.
2.7 Agarose gel electrophoresis
Analysis of DNA was performed using agarose gel electrophoresis (Sambrook et al.,
1982). Horizontal 0.8% – 2% (w/v) TBE agarose slab gels were cast and electrophoresed
49
at 100 V in 0.5 × TBE buffer (40 mM Tris⋅HCl, 1 mM EDTA, 20 mM boric acid, pH 8.5).
Where DNA was to be recovered and used in downstream applications, 0.8% – 2% (w/v)
TA agarose slab gels were cast and electrophoresed at 100 V in 0.5 × TA (20 mM TrisHCl, 10 mM glacial acetic acid, pH 8.5). To allow visualization of the DNA on a UV
transilluminator, the gels were supplemented with 0.5 μg.ml-1 ethidium bromide. The
DNA fragments were sized according to their migration in the gel as compared to that of
standard DNA molecular markers (Lamda DNA restricted with PstI; 100 bp HyperladderI,
Bioline).
2.8 GFX™ DNA purification
Purification of DNA from either solution or agarose gels were performed using the GFX™
DNA and gel band purification kit (Amersham biosciences) according to manufacturer’s
specifications.
2.9 pMOSBlue blunt ended cloning
The pMOSBlue (Amersham Biosciences) cloning plasmid was used in the construction of
the
16S
rDNA
metagenomic
libraries.
pMOSBlue
is
supplied
as
a
blunt
5’
–
dephosphorylated linear plasmid suitable for cloning blunt ended DNA inserts. GFX™purified (Section 2.8) 16S rDNA PCR amplicons were blunt ended, ligated and
transformed into pMOSBlue competent cells according to the manufacturer’s instructions.
2.10 Preparation of electrocompetent E. coli cells
Electrocompetent DH5α, XL1blue and JM109 E. coli cells were prepared as outlined in
Sambrook and Russell (2001), with slight modification. All glassware were thoroughly
50
acid-washed with 30% H2SO4, rinsed and autoclaved prior to use. A single colony of the
E. coli strain was inoculated into 30 ml of LB-broth and incubated at 37°C with shaking
until stationary phase. 10 ml of the culture was transferred to two aliquots of 500 ml of
LB-broth and incubated at 30°C until mid-logarithmic phase (OD600 of 0.4). The flasks
were rapidly cooled in ice-water for 20 min and the cells were collected in polypropylene
tubes by centrifugation at 1000 × g for 10 min in an Eppendorf 5810 R swing bucket
centrifuge. The supernatant was decanted and the cells resuspended in equal volume icecold Millipore water. After harvesting the cells as above, the pellets were resuspended in
250 ml 10% glycerol, collected by centrifugation and the supernatant carefully decanted.
The cell pellet was resuspended in 1 ml GYT medium and the cell density at OD600
adjusted to between 2 × 1010 to 3 × 1010 cells.ml-1. The cells were aliquotted into 40 μl
volumes, and stored at -80°C until required.
2.11 Preparation of chemically competent E. coli cells
Chemically competent cells were prepared according to the method of Hanahan (1983)
with slight modification. All glassware were thoroughly acid-washed with 30% H2SO4,
rinsed and autoclaved prior to use. A single colony of the E. coli strain was inoculated
into 30 ml of LB-broth and incubated at 37°C with shaking until stationary phase. 1 ml of
the culture was transferred to 100 ml of LB-broth and incubated at 30°C until midlogarithmic phase (OD600 of 0.5). The flasks were rapidly cooled in ice-water for 20 min
and 60 ml of the cells were collected in polypropylene tubes by centrifugation at 1000 × g
for 10 min in an Eppendorf 5810 R swing bucket centrifuge. After discarding the
supernatant, the cells were resuspended in 0.5 × volume filter sterilized competency
buffer (0.1 M CaCl2 [w/v], 0.07 M MnCl2 [w/v] and 0.04 M NaOAc [w/v], pH 5.5) and
51
incubated at 4°C for 30 min. Following incubation the cells were harvested by
centrifugation at 1000 × g for 5 min and resuspended in 7.5 ml competency buffer. 575
μl 80% glycerol was added thoroughly mixed and the competent cells dispensed into 100
μl aliquots and stored at -80°C until required.
2.12 Transformation of E. coli cells by:
2.12.1 Electroporation
An Eppendorf tube containing 40 μl of electrocompetent cells was removed from -80°C
and allowed to thaw on ice. 2μl of ligation mix was added to the thawed cells and gently
mixed. The mixture was returned to ice for ~ 1 min then pipetted into a pre-cooled 0.1
cm
sterile
electroporation
cuvette
(Bio-Rad
Laboratories,
Hercules,
CA,
USA).
Electroporation was performed using the following conditions: 1.25 – 1.8 kV, 25 μF, 200
Ω. Immediately following electroporation, 950 μl TB broth, pre-warmed to 37°C, was
added to the cuvette, the cells transferred to a 15 ml Falcon tube and incubated at 37°C
for 1 h with agitation. The cells were plated in aliquots of 5 to 50 μl onto LB-agar plates
supplemented
with
the
appropriate
antibiotic.
Where
applicable,
recombinant
transformants were selected by blue/white colour selection based on insertional
inactivation of the lacZ gene. For this purpose, the cells were spread together with 40 μl
of X-gal (2% [v/v] stock solution) and 10 μl IPTG (100 mM stock solution) over the
surface of LB-agar plates, supplemented with the appropriate antibiotic and incubated
overnight at 37°C.
2.12.2 Chemical
52
An Eppendorf tube containing 100 μl of chemically competent cells was removed from 80°C and allowed to thaw on ice. 2μl of ligation mix was added to the thawed cells and
gently mixed. The mixture was incubated on ice for 30 min then heat-shocked at 42°C for
90 s in a water bath. The Eppendorf tube was returned to ice for 2 min where after 900
μl of sterile LB-broth was added and the Eppendorf tube incubated at 37°C for 1 h with
agitation. The cells were plated in aliquots of 100 to 200 μl onto LB-agar plates
supplemented
with
the
appropriate
antibiotic.
Where
applicable,
recombinant
transformants were selected by blue/white colour selection based on insertional
inactivation of the lacZ gene. For this purpose, the cells were spread together with 40 μl
of X-gal (2% [v/v] stock solution) and 10 μl IPTG (100 mM stock solution) over the
surface of LB-agar plates, supplemented with the appropriate antibiotic and incubated
overnight at 37°C.
2.13 Colony PCR
Following transformation of E. coli cells (Section 2.11), colony PCR was used to screen all
putative recombinant clones. The putative recombinants were aseptically inoculated into
LB-broth containing the appropriate antibiotic (Section 2.1.2) and incubated overnight at
37°C with agitation. Cultures were further analysed by pipetting 200 μl of each into 0.6
ml PCR tubes. The tubes were centrifuged at 13 000 × g to pellet the cells and the
supernatant discarded. The cells were resuspended in 200 μl UHQ Millipore water and
lysed by incubation at 98°C for 5 min. Tubes were then centrifuged at 13 000 × g for 5
min to pellet cell debris. The DNA-containing supernatant served as template in 30 μl
PCR reactions performed essentially as described in Section 2.6 (the annealing
53
temperature was lowered to 49°C). An aliquot of each PCR reaction was analysed by gel
electrophoresis as described in Section 2.7.
2.14 Restriction endonuclease digestion
All restriction enzyme digestions were performed in sterile Eppendorf tubes in small
reaction volumes (10 – 20 μl). The reactions contained the appropriate volume of 10 ×
buffer supplied by the manufacturer for the specific enzyme, and 5 – 10 U of enzyme per
μg of plasmid or genomic DNA. Reactions were incubated for either short periods, 0.5 –
1.5 h, or overnight in a water bath at 37°C, unless specified otherwise. When digestions
included two enzymes requiring different salt concentrations for optimal activity, the
enzyme requiring a lower salt concentration was used first after which the salt
concentration was adjusted and the second enzyme added. The digestion products were
analyzed by gel electrophoresis in 1% or 2% (w/v) agarose gels as described in Section
2.7.
2.15 ARDRA analysis of 16S rDNA amplicons
Following colony PCR (Section 2.12), amplicons of the correct size were subjected to
restriction endonuclease digestion (Section 2.13) by taking 5 μl of the PCR reaction and 6
U of the tetranucleotide-specific enzyme AfaI. Restriction digestions (15 μl) were viewed
on 2% agarose gels following gel electrophoresis (Section 2.7).
54
2.16 Plasmid DNA extraction
2.16.1 Alkaline lysis
Colonies were picked from the agar plates, inoculated into 5 ml of LB-broth
supplemented with the appropriate antibiotic, and incubated overnight at 37°C with
agitation. Plasmid DNA was isolated from the cultures by the alkaline lysis method
(Birnbiom and Doly, 1979), with the following modifications. After incubation, cells from
2 ml of each culture was collected in 2 ml Eppendorf tubes by centrifugation at 10000 × g
for 1 min at room temperature. The supernatant was discarded and the bacterial pellet
suspended in 400 μl of Solution 1 (50 mM glucose, 25 mM Tris⋅HCl, pH 8.0, 10 mM EDTA,
pH 8.0). After incubation at room temperature for 10 min, 400 μl of Solution 2 (1% [w/v]
SDS, 0.2 N NaOH) was added and the tubes were incubated on ice for 10 min. Following
the addition of 300 μl of 7.5 M ammonium acetate (pH 7.6), the tubes were incubated on
ice for 10 min, and then centrifuged at 13 000 × g for 5 min at room temperature. The
plasmid DNA was precipitated from the supernatant by the addition of 650 μl isopropanol
for 10 min at room temperature. The precipitated plasmid DNA was collected at room
temperature by centrifugation at 12 000 × g for 10 min and the supernatant discarded
before addition of 100 μl of 2 M ammonium acetate (pH 7.4). The tubes were incubated
on ice for 10 min. Following ambient centrifugation at 12 000 × g for 5 min, 110 μl of
isopropanol was added to the supernatant and the tubes incubated at room temperature
for 10 min. Precipitated DNA was collected and the pellets washed with 70% ethanol to
remove residual salts from the DNA. The DNA was air-dried and resuspended in UHQ
Millipore water. Plasmid DNA was analyzed on a 1% (w/v) agarose gel as described in
Section 2.7.
55
2.16.2 Talent Kit
Plasmid extractions performed for subsequent nucleotide sequence analysis was
performed using the Talent plasmid purification kit
2.17 Nucleic acid sequencing
Sequencing of cloned insert DNA was performed using the MegaBACE 500 Automated
Capillary DNA Sequencing System (Amersham Biosciences). Where 16S library clone
inserts were sequenced, the internal 16S rDNA E. coli primer F3:16.5, which primes
nucleotides 517 – 536, was used. All other sequencing reactions entailed using universal
vector derived primers M13 forward and/or reverse (Table 1). Cloned 16S rDNA genes
were sequenced by targeting the negative strand allowing 1 × coverage. Both strands of
all the partial lipase genes were sequenced to allow minimum ambiguity.
2.18 Preparation of metagenomic libraries
2.18.1 Metagenomic DNA digestion
Approximately 2 μg aliquots of community DNA were used for restriction enzyme
digestion using AluI. Digestions were performed using 6 U of AluI at time intervals
varying between 45 and 55 min. Following digestion, reaction mixtures and λ-PstI
molecular weight marker were loaded into separate wells of a 1.5% - 0.8% gradient
agarose gel and electrophoresed as described in Section 2.7.
2.18.2 A-tailing of 3’ termini of restriction digested metagenomic DNA
The addition of single adenosine nucleotides to the 3’ ends of the partially digested
metagenomic DNA was performed using a standard PCR reaction. Following restriction
56
endonuclease digestion (Section 2.16) reaction mixtures were used directly in A-tailing
PCR reactions at a concentration not exceeding 30% (v/v). PCR reactions (60 μl)
contained 20 μl restriction digested metagenomic DNA reaction, 1 × NEB PCR buffer (20
mM Tris-HCL (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1% [v/v] Triton X –
100), 0.1 mM dATP, and 1.5 μl Taq DNA polymerase. The PCR reaction mixtures were
placed into an Applied Biosystems thermocycler Gene Amp® 2700 using the following
conditions: 72°C for 30 min followed by rapid cooling to 4°C and gel electrophoresis as
described in Section 2.7.
2.18.3 Recovery of restriction digested metagenomic DNA from agarose
gel
Following electrophoresis and UV visualization of digested A-tailed metagenomic DNA,
the 2 – 10 kb fraction was excised using a sterile scalpel blade. The excised gel was
placed in a sterile 1.5 ml Eppendorf tube and the DNA recovered using the GFX™-gel
extraction kit (Amersham Biosciences).
2.18.4 5’-dephosphorylation of metagenomic DNA
Dephosphorylation of 5’ ends of GFX™-purified genomic DNA was performed using Shrimp
Alkaline Phosphatase (SAP), Roche, according to manufacturer’s specifications. The
reaction was made up to a final volume of 50 μl by adding 12 μl SAP at 1 U.μl-1 and 5 μl
10 × buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 8.5) to the eluted GFX™-purified DNA,
followed by incubation at 37°C for 1 h.
57
2.18.5 Metagenomic library construction
The
pCR®-XL-TOPO®
cloning
kit
(Invitrogen)
was
used
for
the
construction
of
metagenomic libraries. A ligation reaction was set up by combining 5 ng pCR®-XL-TOPO®
and 0.87 μl (130 ng.μl-1) dephosphorylated and A’ – tailed metagenomic DNA.
The
mixture was incubated at 22°C for 5 min and quenched by the addition of 6× TOPO®
cloning stop solution. The ligation reaction was immediately used to transform
electrocompetent E. coli Top10 cells (Invitrogen.)
2.19 Amplification of metagenomic library
After plating 5 μl of the ligation reaction to determine cloning efficiency, the remainder of
the transformation mixture (~995 μl) was removed from 4°C, transferred to 10 ml of LBbroth and grown for 4 – 6 h at 37°C with shaking. After the allotted time, 5 μl of the
culture was plated onto kanamycin selective media and allowed to grow at 37°C over
night. The remainder of culture was stored at 4°C to stunt growth. The following day, the
colonies were counted and the theoretical number of times the original library was
amplified determined. Colonies counted following plating after 4 h of shaking at 37°C
were eight times more compared to t=0. To the ~ 11 ml of culture, 11 ml of 30% sterile
glycerol was added and thoroughly mixed. Aliquots of 2.75 ml, which represented a
single copy of the library, was made into labeled 2 ml sterile screw-cap tubes and stored
at -80°C until needed.
2.20 Activity-based screening of the metagenomic library
Two copies of the library were thawed on ice and separately plated in aliquots of 60 μl
(~2000 clones) onto esterase and lipase specific media and incubated at 37°C until
58
colonies were visible. The plates were removed and incubated further at 16°C for 7 d and
monitored for the presence of lipolytic activity.
2.21 PCR-based screening of the metagenomic library
A single copy of the library was subjected to alkaline lysis to purify the recombinant
pCR®-XL-TOPO® plasmid DNA. The isolated plasmid DNA was divided into ten aliquots and
used as template DNA for the detection of lipolytic genes during PCR. A neat, 1:10, 1:20
and 1:50 dilution was prepared from each aliquot and each template was added to a
separate PCR reaction mix (30 μl) containing 1 × NEB PCR buffer (20 mM Tris-HCL (pH
8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1% [v/v] Triton X – 100), 0.1 mM
dATP, 0.5 pmol of each primer LipF and LipR2 and 1.5 μl Taq DNA polymerase. PCR
reaction mixtures were placed in an Applied Biosystems thermocycler Gene Amp® 2700
using the following PCR cycling conditions: Initial denaturation at 95°C for 90 s, followed
by 35 cycles of denaturation at 94°C for 20 s, annealing at 50°C for 40 s, and extension
at 72°C for 1 min. The final elongation step was performed at 72°C for 3 min. For control
purposes, a reaction mixture containing all reagents except template was included. A
positive reaction containing a cloned lipase gene of B. multivora as template DNA was
included. An aliquot of each reaction mixture was analysed using gel electrophoresis as
described in Section 2.7
2.22 PCR amplification of partial lipase fragments
For amplification of partial lipase fragments, primers LipF and LipR2 (Table 1) were used.
Community DNA isolated from Antarctic Dry Valley mineral soils served as template for
reactions. The PCR reactions (20 – 30 μl) contained between 10 and 50 ng genomic DNA,
59
1 × NEB PCR buffer (20 mM Tris-HCL (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM
MgSO4, 1% [v/v] Triton X – 100), 2 mM MgCl2, 0.2 mM of each deoxynucleoside
triphosphate (dNTP), 0.5 pMol of each primer and 1 U Taq polymerase. The PCR
reactions were placed in a Perkin-Elmer 2400 thermocycler using the following
conditions: initial denaturation at 94°C for 5 min, followed by 4 cycles of denaturation at
94°C for 30 s, annealing at 65°C for 1 min, and elongation at 72°C for 2 min. Cycling
conditions were then altered: the same denaturation and elongation conditions were
used, but the annealing temperature was reduced to 64°C with a reduction of 1°C every
cycle for 14 cycles. Cycling conditions were again adjusted: the denaturation and
elongation conditions were maintained, but the annealing temperature was set at 50°C
for 20 cycles. Following the last cycle, a final elongation step at 72°C for 3 min was
performed. For control purposes, a reaction mixture containing all reagents except
template DNA was included. Aliquots of the PCR reaction mixtures were subsequently
analyzed by agarose gel electrophoresis as described in Section 2.7.
60
Chapter 3
Bacterial diversity in Antarctic Dry Valley mineral soils
3.1 Introduction
Temperate and tropical soil communities are regarded as among the most complex and
diverse assemblages of microorganisms (Kuske et al., 1997), with estimated bacterial
numbers in the order of 109 cells.g-1 and over 104 distinct species, as shown by
reassociation kinetics (Dunbar et al., 2002; Torsvik et al., 1990). However, the
desiccated mineral soils of the Dry Valleys, Ross Dependency, Eastern Antarctica are
generally thought to harbour very low cell densities (Cameron et al., 1972) supporting
the perception that these so-called extreme environments exhibit low species diversity
and low cell numbers. This is attributed to the imposition of environmental extremes
which, for the Antarctic Dry Valleys, include low temperatures, wide temperature
fluctuations, low nutrient status, low water availability, high incident radiation and
physical disturbance (Wynn-Williams, 1990). Nevertheless, is was recently shown by
ATP, lipid and DNA quantitation that Dry Valley mineral gravels may contain between 106
and 108 prokaryotic cells.g-1 (Cowan et al., 2002).
The current understanding of Antarctic mineral soil microbiology is based almost
exclusively on culture-based studies. These studies have suggested that most Antarctic
microbes belong to a restricted number of cosmopolitan taxa and are largely aerobic,
with only few reported anaerobic isolates (Friedmann, 1993). Large numbers of
coryneform-related
bacteria
such
as
Arthrobacter,
61
Brevibacterium,
Cellulomonas,
Corynebacterium were reported together with gracilicutean isolates (members of the
Gram-negative Eubacteria) such as Pseudomonas and Flavobacterium. Firmicutean
bacteria isolated included Bacillus, Micrococcus, Nocardia, Streptomyces, Flavobacterium
and pseudomonads (Cameron et al., 1972; Friedmann, 1993). A number of less common
genera such as Beijerinckia, which rarely occur outside tropical soils, Xanthomonas, a
pathogen associated with higher plants and Planococcus, a marine genus, have also been
isolated from Antarctic soils (Friedmann, 1993). Cyanobacteria are also well-documented
inhabitants of Antarctic soil biotopes (de la Torre et al., 2003; Taton et al., 2003) but are
thought to be restricted to moist habitats (de los Ríos et al., 2004).
It is now widely acknowledged that culture-based community studies inevitably induce a
high degree of bias, while important groups of organisms which may be fastidious, coculture-dependant or in a viable but non-culturable (VBNC) state may be unrepresented
(Amann et al., 1995; Holmes et al., 2000; McDougald, 1998; Waterbury et al., 1979). It
is therefore probable that historical data from culture-dependant studies do not
accurately represent the true microbial species diversity of the Dry Valley mineral soils. A
number of important Antarctic Dry Valley microbial biotopes, including cryptoendolithic
communities (de la Torre et al., 2003), cryoconite holes (Christner et al., 2003), and lake
ice and marine ice flows (Priscu et al., 1998) have been subject to detailed community
analyses using modern molecular phylogenetic techniques. However, the supposedly less
complex and more ‘extreme’ mineral soils have yet to be investigated in detail.
62
3.2 Aim
The principle aim of the work reported in this chapter was to assess and compare
bacterial diversity of three different Antarctic Dry Valley mineral soils.
To achieve this aim, the following objectives were set forth:
)
Isolation of pure, intact high molecular weight DNA from all three sample sites
)
PCR amplification of 16S rDNA genes
)
Construction of three independent 16S rDNA libraries
)
Sequence analysis and identification of unique 16S clones
3.3 Results
3.3.1 Soil properties
The properties for each of the soil samples used in this study are listed in Table 3.1.
Sample MVG was a fine particulate gravel collected from the surface on a south-facing
slope 225 m above sea level. This mid-altitude sample represents a desiccated,
oligotrophic habitat, exposed to the harsh environmental conditions (Wynn-Williams,
1990). DNA yields from multiple extractions averaged 840 ng.g-1.
Sample PENP was recovered in a saddle between Miers and Shangri-La Valleys at an
altitude of 585 m above sea level. The site was approximately 50 m from the margin of
Lake Purgatory. PENP is also desiccated and oligotrophic but likely to be subject to a
higher incidence of humidity due to the altitude.
63
Table 3.1
Site descriptions for Dry Valley mineral soil samples
Sample
Site description
GPS
code
PENP
Dry sand from high
altitude site
MVG
Dry sorted sands and
gravels from mid
78o 04.762’
DNA
Protein
Lipid
Altitude
(H2O)
(ng/g)
(mg/g)
(μg/g)
(m)
(% wt.)
480
280
86
584
0.7
840
386
66
225
0.8
320
656
326
2
6.2
o
165 52.083’
78o 06.140’
o
165 48.646’
altitude valley slopes
BIS
Fine dark particulate
soil from underside of
78o 00.966’
o
165 32.795
seal carcass
Sample BIS was a eutrophic soil collected from the underside of a crab-eater seal
carcass. The soil was dark and had a clay-like consistency. BIS showed high levels of
both protein and lipid and contained almost 8-fold more water than the other two sites. A
higher water content implies the presence of organic matter, as organic matter has been
shown to facilitate water retention (Smith and Tearle, 1985). Higher levels of lipid and
protein might be attributed to the high levels of organic carbon which provide nutrition
facilitating a higher turnover. Higher turnover of nutrients and increased biological
activity could possibly explain the lower extracellular DNA levels noted for BIS compared
to MVG and PENP. Not only were the DNA yield from BIS low, but also contained high
levels of humic acids and other PCR inhibitors. Higher DNA yields obtained from MVG and
PENP might be due to low turnover within the soils. DNA isolated from MVG and PENP
also showed no humic acid contamination requiring no additional purification prior to
PCR. DNA quantities isolated from the mineral soils however were significantly lower
compared to DNA yields of between 2 and 50 μg.g-1 from more temperate soils (Stach et
al., 2001).
64
3.3.2 Cloning of the 16S rDNA PCR-amplicons into pMOSBlue
To assess the prokaryotic diversity in the Antarctic cold mineral deserts, universal
primers E9F (Farelly et al., 1995) and U1510R (Reysenbach and Pace, 1995) were used
to target and PCR-amplify 16S rDNA genes. The primers were designed to target the
conserved regions at the 3’ and 5’ ends of the 16S gene, and to yield a 1.5 kb PCR
product. Using community DNA isolated from all three sample sites as template, PCR was
performed as described is Section 2.6 (Fig. 3.1). A single amplicon of 1.5 kb was
observed following gel electrophoresis (Fig. 3.2). No amplification products were
observed in the negative control. The gel-purified amplicons were polished and
phosphorylated as described in Section 2.9 and ligated to the pMOSBlue cloning plasmid.
Following transformation of MOSBlue competent cells, recombinant transformants with a
Gal- phenotype on X-gal containing indicator plates were subjected to colony PCR
(Section 2.12). Colonies yielding 1.5 kb PCR-amplicons (Fig. 3.2) using vector-derived
M13 forward and M13 reverse primers were subjected to ARDRA analysis. False positives
which failed to yield amplicons or produced amplicons of the incorrect size (Fig. 3.3 lanes
2 and 10) were discarded.
3.3.3 16S rDNA ARDRA analysis
Recombinant pMOSBlue plasmids containing the expected 1.5 kb 16S rDNA sequences
were subjected to ARDRA. The number and size of each DNA fragments signified a
unique ARDRA pattern (Fig. 3.4). ARDRA analyses were continued until 20 unique 16S
rDNA clones from each sample site had been identified. In total, 181 clones were
analysed, 81 from PENP, 55 from MVG and 45 from BIS. This implies that 24% of PENP,
36% of MVG and 40% of BIS clones were unique. Collector’s curve data (Fig. 3.5)
65
1
2
3
4
14 kb
Fig. 3.1
Metagenomic DNA extracted from Dry Valley mineral soils prior to 16S PCR. Lane 1,
DNA molecular weight marker; Lane 2, MVG metagenomic DNA; Lane 3, PENP
metagenomic DNA, Lane 4, BIS metagenomic DNA. The sizes of the DNA molecular
weight marker, phage lambda DNA digested with PstI, are indicated to the left of
the figure.
1
2
3
4
5
1.7 kb
1.08 kb
Fig. 3.2
Amplicons obtained following 16S PCR amplification using MVG, PENP and BIS
metagenomic DNA as template. Lane 1, DNA molecular marker; Lane 2, Sample of
the PCR reaction using MVG as template; Lane 3, Sample of the PCR reaction using
PENP as template; Lane 4, Sample of the PCR reaction using BIS as template; Lane
5, Negative control lacking template DNA. The sizes of the DNA molecular weight
marker, phage lambda DNA digested with PstI, are indicated to the left of the
figure.
66
1
2
3
4
5
6
7
8
9
10
11
12
1.7 kb
1.09 kb
Fig. 3.3
Amplicons obtained following colony PCR amplification of the 16S rDNA genes using
plasmid pMOSBlue DNA as template. Lane 1, DNA molecular weight marker; Lanes
2 – 11, Sample of reaction mixture following PCR; Lane 12, Negative control
reaction lacking template DNA. The sizes of the DNA molecular weight marker,
phage lambda DNA digested with PstI, are indicated to the left of the figure.
1
2
3
4
5
6
7
8
9
800 bp
540 bp
340 bp
200 bp
Fig. 3.4
16S rDNA amplicons subjected to ARDRA analysis. Lane 1, DNA molecular weight
marker; Lanes 2-9, 16S rDNA amplicons restricted with AfaI. The sizes of the DNA
molecular weight marker, phage lambda DNA digested with PstI, are indicated to
the left of the figure.
67
25
Unique clones
20
PENP
15
10
MVG
5
BIS
81
76
71
66
61
56
51
46
41
36
31
26
21
16
6
11
1
0
Total number of clones
Fig. 3.5 Collectors curves of clone culture libraries MVG, PENP and BIS.
indicates that all three samples are still rising and that sampling has not been exhausted.
3.3.4 16S rDNA analysis and distribution
Partial sequence data was obtained using internal primer F3:S16.5 (Table 2.1).
Sequences obtained and used for BLASTn analysis included variable regions V5 and V6
and part of V4 (Fig. 1.1). Bacteria identified from the 60 sequences representing all 181
clones grouped into eight broad phylogenetic groups (Cyanobacteria, Actinobacteria,
Acidobacteria,
Verrucomicrobia,
α-Proteobacteria,
β-Proteobacteria,
Chloroflexi
and
Bacteroidetes) (Table 3.2) and showed an average identity of ≥91% to known
phylotypes. Three phylotypic groups showed distribution in all three sites where the
remaining five were either unique to one or two sites. Cyanobacteria (13%),
Actinobacteria (26%) and Acidobacteria (16%) represented the majority of the identified
phylotypes.
68
3.4 Discussion
3.4.1 Phylotype coverage
Cyanobacteria
are
generally
associated
with
moist
environments
making
water
availability the principal factor dictating cyanobacterial distribution. For example,
cyanobacteria are common in moist soils (Miller and Bebout, 2004) and aquatic habitats
including glacial streams (Vincent et al., 1993) and flushes (Horne, 1972), saline lakes
(Parker and Wharton, 1985) and cryoconite holes (Christner et al., 2003). Cyanobacteria
are known to form thick microbial mats on the upper surface of lake sediments (Broady,
1996). The ability to spread out on various surfaces is conferred by gliding motility of
filamentous cyanobacteria together with the secretion of extracellular polysaccharides
(EPS). This results in a highly pigmented and structured biofilm covering the substrate
(de los Ríos et al., 2004). Their presence in the PENP sample might be indicative of
widespread occurrence in the Dry Valleys and their phototrophic ability might contribute
to an active community structure.
Acidobacteria is a newly recognized group of microorganisms widespread in soil biotopes.
They show diverse physical and chemical properties (Barns et al., 1999) and form a
major fraction of non-cultured bacteria (Stevenson et al., 2004). There are only three
cultured representatives: Acidobacterium capsulatum, Halophaga foetida and Geothrix
fermantans. A. capsulatum is a moderately acidophilic aerobic heterotroph (Hiraishi et
al., 1995) whereas H. foetida and G. fermentans are strict anaerobes that ferment
aromatic compounds and acetate, respectively (Lonergan et al., 1996; Liesack et al.,
1994). The majority of sequences that make up this division are from environmental
clones. In fact, of the eight monophyletic subdivisions of the Acidobacteria, subdivisions
69
1, 3, 4 and 6 are represented by environmental clone sequences only (Hugenholtz et al.,
1998). The widespread occurrence of members of the Acidobacteria division detected in
environmental samples suggests their presence might be ecologically significant. They
have been detected in peat bog, acid mine drainage, hot spring, and fresh water lake
systems (Stevenson et al., 2004; Barns et al., 1999; Hiraishi et al., 1995)
Actinobacteria are high GC rich Gram-positive hetrotrophic bacteria (Basilio et al., 2003).
They are one of the better-defined phylogenetic groups and well represented in culture
studies. However, a small number of genera are poorly known and are so highly
divergent from all the other members of the group that they classify as subclasses
(Stackebrandt et al., 1997). These subclasses are small, each represented by a single or
a very few strains, and include Rubrobacteridae, Acidimicrobidae, Sphaerobacteridae and
Coriobacteridae (Holmes et al., 2000). Rubrobacteria are abundant in a variety of
environments and have been documented in agricultural soil (Ueda et al., 1995), peat
(Rheims et al., 1996), pasture soil (McCaig et al., 1999) and forest soil (Liesack and
Stackebrandt, 1992).
Verrucomicrobia is a newly proposed division of Bacteria represented by a small fraction
of
isolates:
Verrucomicrobium
spinosum
(Ward-Rainey
et
al.,
1995),
four
Prosthecobacter species (Hedlund et al., 1997) and three strains of ultramicrobacteria
(Janssen et al., 1997). Verrucomicrobia and Prosthecobacter were isolated from fresh
water and the ultramicrobacteria were isolated from soil. All of the isolates preferentially
use sugars as growth substrates and, like members of the Acidobacteria, are widespread
and abundant in the environment, especially soil (Hugenholtz et al., 1998). Of the
70
several monophyletic subdivisions, only two represent cultured isolates. Interestingly,
four species of the Verrucomicrobial genus Prosthecobacter might constitute an
evolutionary link between the members of Bacteria and Eucarya due to the presence of
genes for tubulin production, a cytoskeletal element previously only found in eukaryotes
(Jenkins et al., 2002; Staley et al., 2005)
3.4.2 Phylotype distribution
Most prominent within the PENP clone set were two orders of cyanobacteria;
Oscillatoriales and Nostocales. Clone PENP49 showed close relatedness (98%) to Nostoc
sp. and PENP18 to Phormidium sp. Ant-lunch (99%). Clone PENP4 showed similarity to
the Oscilliatorium, Phormidium tenue.
The majority (25%) of the BIS clones grouped with uncultured environmental bacteria
(Table 3.2). Nevertheless, the uncultured clones were phylogenetically diverse and
primarily mapped to the actinobacteria, acidobacteria and α-protebacteria. Three of the
BIS clones, BIS 31 grouped with unclassified bacteria, rendering their taxonomical status
undefined.
Cyanobacteria appeared to be restricted to only the high altitude PENP sample site. The
appearance of cyanobacterial phylotypes as major contributors to only one of the three
clone libraries supports the consensus that cyanobacterial distribution in the Dry Valleys
is non-homogeneous (Vishniac, 1993).
71
Table 3.2
Unique 16S rDNA clone sequences identified in three different Antarctic mineral soils
Sample site/clones
(Accession number)
Phylogenetic group
I.D. of nearest match
(Accession number)
%
ID
PENP4 (DQ062859)
PENP7 (DQ062860)
PENP18 (DQ062861)
PENP22 (DQ062862)
Cyanobacteria
Actinobacteria
Cyanobacteria
Cyanobacteria
96
99
99
97
PENP25 (DQ062863)
Cyanobacteria
PENP35 (DQ062864)
PENP37 (DQ062865)
PENP40 (DQ062866)
Environmental samples
Bacteroidetes
Actinobacteria
PENP42 (DQ062867)
PENP48 (DQ062868)
Bacteroidetes
Verrucomicrobia
PENP49 (DQ062869)
PENP50 (DQ062870)
Cyanobacteria
Bacteria, environmental
sample
Actinobacteria
Bacteroidetes
Leptolyngbya sp. (AY239604)
Arthrobacter agilis (AF134184)
Phormidium sp. Ant-Lunch (AF263335)
Uncultured Antarctic bacterium
(AF076163)
Uncultured Antarctic Cyanobacterium
(AY151721)
Uncultured organism clone (AY897885)
Uncultured Bacteroidetes (AY689627)
Uncultured Actinobacterium clone
FBP460 (AY250884)
Uncultured bacterial clone (AJ290025)
Uncultured soil bacterial clone C019
(AF013522)
Nostoc sp. (AY566855)
Uncultured bacterial clone csbio160368
(AY187335)
Uncultured Actinobacterium (AY690206)
Uncultured Bacteroidetes clone VC5
(AY211071)
Uncultured Acidobacterium (AY571794)
Uncultured bacterium (AY662047)
Uncultured bacterial clone sipK9
(AJ307936)
Oscillatoria sp. Ant-G16 (AF26333)
Trichormus azollae (AJ630454)
Uncultured Antarctic cyanobacterium
(AY151722)
Geodermatophilus sp. G1S (X92364)
Uncultured bacterial clone C-F-15
(AF443586)
Uncultured Actinobacterium (AY690226)
Uncultured bacterial clone CO19
(AF013522)
Bacterium Ellin504 (AY960767)
Conexibacter woesei (AJ440237)
Uncultured Acidobacterium (AY571792)
Uncultured bacterial clone FBP241
(AY250867)
Uncultured bacterial clone SO27
(AF013554)
Uncultured bacterial clone D132
(AY274138)
Uncultured Acidobacterium (AY571794)
Uncultured Actinobacterium (AF234135)
Uncultured bacterium (AY922044)
Uncultured Acidobacterial clone BAC220H (AY214900)
Uncultured Bacteroidetes (AY921683)
Actinobacterial strain PB90-4
(AJ229240)
93
PENP54 (DQ062871)
PENP55 (DQ062872)
PENP62 (DQ062873)
PENP68 (DQ062874)
PENP75 (DQ062875)
PENP76 (DQ062876)
PENP77 (DQ062877)
PENP78 (DQ062878)
MVG1 (DQ062879)
MVG2 (DQ062880)
MVG5 (DQ062881)
MVG9 (DQ062882)
MVG14
MVG15
MVG16
MVG18
(DQ062883)
(DQ062884)
(DQ062885)
(DQ062886)
MVG19 (DQ062887)
MVG20 (DQ062888)
MVG21
MVG23
MVG24
MVG25
(DQ062889)
(DQ062890)
(DQ062891)
(DQ062892)
MVG28 (DQ062893)
MVG31 (DQ062894)
Acidobacteria
Bacteria, environmental
samples
Bacteria, environmental
sample
Cyanobacteria
Cyanobacteria
Cyanobacteria
Actinobacteria
Bacteria, environmental
sample
Actinobacteria
Verrucomicrobia
Actinobacteria
Actinobacteria
Acidobacteria
Bacteria, environmental
sample
Bacteria, environmental
sample
Bacteria, environmental
sample
Acidobacteria
Actinobacteria
Chloroflexi
Acidobacteria
Bacteriodetes
Actinobacteria
72
95
93
97
99
96
95
98
98
92
96
92
93
99
95
97
94
96
94
96
95
96
98
95
96
95
94
94
98
92
96
96
Table 3.2 continued
Sample site/clones
(Accession number)
Phylogenetic group
I.D. of nearest match
(Accession number)
%
ID
MVG50 (DQ062895)
MVG51 (DQ062896)
MVG52 (DQ062897)
Actinobacteria
Actinobacteria
Unclassified,
environmental sample
Bacteria, environmental
samples
Actinobacteria
Kribella sp. (AJ811962)
Bacterium Ellin301 (AF498683)
Uncultured organism (AY897921)
95
95
94
Uncultured bacteria candidate division
OP10 (AY192276)
Intrasporangium calvum DSM 43043T
(AJ566282)
Uncultured Actinobacterium (AY250884)
Uncultured soil bacterial clone CO133
(AF507702)
Uncultured Flavobacterium sp.
(AY571822)
Uncultured β-proteobacterium
(AY690290)
Uncultured α-proteobacterium
(AJ532707)
Uncultured Acidobacterium (AY571792)
Uncultured Actinobacterium (AF498707)
Uncultured Acidobacterium (AY281358)
Uncultured Acidobacterium (AY921997)
Uncultured Acidobacterium (AY921984)
Agricultural soil bacterial clone SC-I-54
(AJ252640)
Uncultured bacterium (AJ1863268)
92
Uncultured bacterial clone D130
(AY274136)
Uncultured Acidobacterium (AY214900)
Uncultured soil bacterium (AY493980)
95
Uncultured Rubrobacter sp. (AY571811)
Uncultured Rubrobacterium (AY395449)
Uncultured bacterial clone W1-4H
(AY192276)
Uncultured bacterium #0319-23B10
(AF234122)
98
91
92
MVG54 (DQ062898)
BIS1 (DQ062899)
BIS2 (DQ062900)
BIS5 (DQ062901)
BIS6 (DQ062902)
Actinobacteria
Bacteria, environmental
samples
Bacteroidetes
BIS8 (DQ062903)
β-proteobacteria
BIS10 (DQ062904)
α-proteobacteria
BIS12
BIS13
BIS15
BIS16
BIS18
BIS20
Acidobacteria
Actinobacteria
Acidobacteria
Acidobacteria
Acidobacteria
Bacteria, environmental
sample
Bacteria Environmental
samples
Bacteria Environmental
samples
Acidobacteria
Bacteria, environmental
samples
Actinobacteria
Actinobacteria
Bacteria, candidate
division OP10
Chloroflexus
(DQ062905)
(DQ062906)
(DQ062907)
(DQ062908)
(DQ062909)
(DQ062910)
BIS21 (DQ062911)
BIS23 (DQ062912)
BIS25 (DQ062913)
BIS26 (DQ062914)
BIS28 (DQ062915)
BIS29 (DQ062916)
BIS31 (DQ062917)
BIS45 (DQ062918)
97
96
91
96
95
95
98
96
95
92
91
93
98
91
91
91
There was no visible evidence of an adjacent cyanobacterial source (either algal mat
material or hypolithic sites) at the time of sampling. The cyanobacterial phylotypes
identified in sample PENP (putatively members of the genera Nostoc, Phormidium and
Oscillatoria) are also not indicative of cryptoendolithic origin. The cyanobacterial
components of these communities are principally members of the Gloeocapsa, Anabaena,
Chroococcidiopsis, Hemichloris, Heterococcus and Lyngbya (Nienow and Friedmann,
73
1993). It is also notable that the hills on the northern flanks of the Miers Valleys appear
to lack the sandstone and marble rock strata that principally harbour cryptoendolithic
communities (Nienow and Friedmann, 1993). It is thus suggested that the cyanobacterial
signals identified in sample PENP constitute free-living cyanobacteria. Although the PENP
sample showed a very low water content (0.7% wt. H2O) as determined by dry weight
measurements, the data might suggest that such values are a poor determinant of
cyanobacterial distribution. Both meteorological studies (Wynn-Williams, 1990) and
surveys of lichen distribution (Pannewitz et al., 2003) indicate that water availability is
strongly altitude dependent. Relative humidity measurements from altitudinal transects
in the Taylor and Wright Valleys (Horowitz et al., 1972) suggest that atmospheric
humidity may be an important determinant of water availability. However, a detailed
survey of cyanobacterial distribution in relation to environmental and physical factors
(altitude, soil type, soil water content, soil humidity, and atmospheric humidity) is
required to more fully understand the factors that dictate the distribution of soil-borne
cyanobacterial populations.
Actinobacterial signals were frequent in all three sites, a result that is consistent with the
widespread distribution of actinobacteria in various soil types (Basilio et al., 2003). The
high frequency of actinobacterial phylotype signals (Table 2) suggests that this group
contributes a significant fraction of the soil microbial population, as shown by others
(Holmes et al., 2000). However, few of the actinobacterial sequences could be matched
to known phylotypes at >95% homology, suggesting that a substantial pool of novel
uncultured psychrotrophic actinobacterial species remain to be identified. Given the
industrial importance of this group of organisms (Bunch, 1998; Jorgensen et al., 2001),
74
this observation adds some weight to the importance of developing new isolation
strategies (Janssen et al., 2002).
The acidobacterial signals were the most highly populated clade in the nutrient-rich BIS
sample (Fig 3.5). 18% of all phylotypic signals obtained were assigned as ‘uncultured’,
and were prevalent in all three sites (Fig 3.5). Phylotypic signals identified as members
of the acidobacteria were major components of both the desiccated mid-altitude (MVG)
and C-enriched (BIS) samples.
While PCR-based analyses of microbial diversity are widely acknowledged to be less than
fully representative due to biases introduced by factors such as extraction efficiency
(Farelly et al., 1995), and hybridisation specificity (Chandler et al., 1997; Suzuki and
Gionvannoni, 1996) it is also accepted that community composition is more effectively
elucidated by this method than by conventional culturing. The results indicate that a
diverse range of prokaryote phylotypes is present in Antarctic Dry Valley cold desert
mineral soils. It was noted, as have others (Lipson and Schmidt, 2004), that the highest
proportion of sequences identified fall into the so-called ‘unculturable’ class. A significant
proportion of the sequences obtained showed relatively low homology to extant
sequences (<95%), suggesting that the mineral soils of the Dry Valleys represent a
substantial pool of novel species and/or genera. Calculations of diversity indices such as
the Shannon-Weiner index support previous suggestions that all three sample sites
harbour relatively low species diversity (Table 3.3) (Mckay, 1993; Vincent, 1988). The
calculated values, H = 1.598, 1.331, and 1.238 for PENP, MVG, and BIS respectively, are
substantially lower than would be expected for temperate soil biotopes, which typically
75
have values of between 6 and 7 (Dunbar et al., 2000; Hughes et al., 2001). Evenness (E)
values calculated are very low for all three samples. At an optimum even distribution, a
value of 1 is given, which implies that the mineral soils indicate a rather high degree of
unevenness (Zar, 1999). The estimated species richness (S) for all thee samples is low
(Table 3.3), supporting the consensus that extreme environments exhibit low species
diversity (Sandaa et al., 1999). Accurately determining species richness is also hindered
by the fact that large numbers of the phylotypes are not identifiable to species level and
that the collectors curves have not reached a plateau (Schloss and Handelsman, 2004).
Table 3.3
Comparison of phylotype richness, diversity and evenness values for the Antarctic
mineral soil PENP, MVG and BIS bacterial communities.
16S rDNA clone libraries
Index
PENP
MVG
BIS
a
49
50
57
b
1.598
1.331
1.238
c
0.410
0.340
0.306
S
H
E
a
Phylotype richness, S, was calculated as the percentage of the total number of distinct ARDRA
patterns to clones.
b
Shannon-Weiner diversity index (Dunbar et al., 2000) was calculated as follows: H = -∑(ρί)(log2
ρί), where ρ is the proportion of an unique ARDRA pattern relative to the sum of all patterns.
c
Evenness (Dunbar et al., 2000) was calculated from the Shannon-Weiner diversity function as
follows: E = H/Hmax where Hmax = Log2(S)
Richness as indicated by the collection curve (Fig. 3.5) equals twenty, since sampling
was continued until only twenty unique clones had been identified for each of the three
soil
samples.
Additional
sampling
should
increase
phylotype
approximated values for species richness in Table 3.3 are reached.
76
richness
until
the
5%
5%
5%
5%
5%
20%
5%
30%
15%
25%
5%
40%
5%
20%
5%
15%
5%
40%
5%
15%
PENP
Fig 3.6
MVG
25%
BIS
Relative percentage distribution of the phylotypes identified from the Antarctic Dry Valley mineral soils.
77
Cyanobacteria and actinobacteria, two of the dominant phyla identified in the oligotrophic
mineral soil samples (MVG and PENP), are well represented in early culture-dependent
studies of Antarctic Dry Valley mineral soil microbiology (Cameron et al., 1972).
However, readily isolatable taxa, such as Achromobacter, Bacillus, Corynebacterium,
Micrococcus, Planococcus and Pseudomonas (Cameron et al., 1972), are not represented
in any of the three 16S rDNA clone libraries. Conversely, groups such as Acidobacteria,
Verricumicrobia and Bacteroidetes, which are absent from both historical and recent
culture-dependent studies appear to be relatively common on the basis of 16S clone
distribution. These groups have also been identified as significant components of the
microbial communities in two previous molecular phylogenetic studies of specific
Antarctic habitats: the cryptoendolithic (de la Torre et al., 2003) and cryoconite hole
communities (Christner et al., 2003).
It has not been established whether the desiccated mineral soils of the Dry Valleys of
Antarctica constitute stable microbial communities, or merely assemblages of organisms
attached to mobile particulates. The mobility of the mineral soils of the Dry Valleys
(particularly during the windy austral winter seasons (Wynn-Williams, 1990) argues for
the latter. A stable community is presumed to contain the elements of energy capture
and turnover of primary nutrient components. Two of the three sites analysed in this
study may thus represent putative communities: the phototrophic and N2-fixation
capacity of the cyanobacterial phylotypes identified in sample PENP, and the presence of
an exogenous nutrient source in sample BIS offer the elements required to maintain a
trophic structure. This cannot be said of the desiccated mineral soils of much of the Dry
Valleys (represented by sample MVG in this study). Although certain phylotypic groups
78
identified in sample MVG are known to include chemoautotrophic species (e.g., the βproteobacteria (Hoeft et al., 2004; Sinigalliano et al., 2001)) the presence of specific
chemoautotrophic
species
cannot
be
directly
inferred
from
these
results.
The
demonstration of a community structure may nevertheless be inferred by identification of
both the presence and activity of key genes, enzymes and/or processes. One possible
way to demonstrate metabolically active communities is the isolation of environmental
RNA. RNA is an ideal biomarker because it serves as a function of DNA copy number and
has a high turnover and would serve as a good indicator of cellular activity (Manefield et
al., 2002). RNA isolation is, however only indicative of the genes expressed at a given
time and does not provide information regarding the organism of origin. More focused
analysis of metabolically active communities could involve stable-isotope-labeled (SIP)
substrates (Pelz et al., 2001). With this approach specific genes and organisms involved
in the assimilation of the chosen substrates can be identified (Boschker and Middelburg,
2002).
16S rDNA analysis performed on the extracted metagenomic DNA from all three sample
sites allowed the documentation of several phylotypes (Fig. 3.6). The probability of
having isolated DNA only from live and/or active cells needs some consideration. It is
likely that the DNA isolated for 16S PCR analysis might also have originated from sources
other than metabolically active cells and could include DNA from VNBC individuals,
extracellular DNA (naked DNA) and randomly wind-dispersed cells or particles. Methods
typically applied to extract DNA from environmental soil samples are designed to extract
total DNA (extra- and intracellular) although robust methods such as bead beating
lessens both quality and quantity of the extracellular DNA isolated compared to more
79
gentle chemical extraction procedures (England et al., 2004). Upon cell death and lysis,
DNA is released into the environment and becomes available to bacterial nucleases.
Despite the presence of nucleases, naked DNA persists in soil for varying lengths of time
(Recorbet et al., 1993). Exogenous DNA is stabilized through the formation of DNA-soil
particle complexes by adsorbing to negatively charged soil and organic matter forming
cation bridges that shield DNA from degradation (Lorenz et al., 1994). Other factors
conducive to the preservation of naked DNA within the Antarctic mineral soils are the
extreme cold (especially in the permafrost layer (Stokstad, 2003)), low water availability
and high levels of salts. The persistence of naked DNA in soil has been demonstrated in
both temperate (forest soils, sediments and caves) and Arctic soils (Stokstad, 2003).
Naked DNA survival was demonstrated by seeding Dry Valley mineral soil with S.
epidermidis genomic DNA, which remained detectable for 23.5 weeks (Ah Tow and
Cowan, 2005). England and co-workers (2004) demonstrated naked DNA survived for
three months in forest soils and long-term survival of naked DNA was indicated by
recovering plant and animal DNA from Siberian sediments dating back to 400 000 and 30
000 years, respectively, (Stokstad, 2003).
If the survival of naked DNA within Antarctic soils is as persantant as in other cold
environments such as the Siberian sediments, it is likely that the 16S rDNA signals
observed in this study might be from both active cells and from naked DNA. The
presence of culturable organisms has been shown by previous culture-based analysis of
the mineral soils (Friedmann, 1993). The possibility that naked DNA from 16S rDNA
sequences has been cloned can not be ruled out, which indicates the necessity for further
investigation to determine the ratio, if any, of naked DNA to culturable cells.
80
The phylotypes identified within this study of Antarctic mineral soils depict preliminary
findings, and additional sampling at various distance intervals from the original sample
sites would give a better indication as to the phylotype composition and richness within
the mineral soils. Sampling at different transects and if possible at intervals throughout
the year will also yield valuable information regarding the distribution of the phylotypes.
Dominant and indigenous phylotypes to each mineral soil should be perpetual throughout
the year whereas exogenous phylotypes, for instance wind-blown individuals or
communities, would be periodic.
Use of SIP (Pelz et al., 2001) and BrdU (Borneman, 1999) would indicate active
communities within the soils. This would aid in differentiating between those phylotypes
forming part of the active bacterial community and derived from dead cells and/or
extracellular DNA.
81
Chapter 4
Metagenomic library construction
4.1 Introduction
The cloning of microbial DNA directly from the environment to screen for the presence of
desired enzyme activity has become a useful tool in discovering new biocatalysts. DNA
libraries have been constructed from various diverse environmental regions such as the
Arctic, hotsprings, deserts and both fresh and sea water (Cottrell et al., 1999; Lee et al.,
2004; Kim et al., 2006). The libraries contain the collective microbial DNA complement,
referred to as the metagenome (Handelsman et al., 1998), of each specific environment.
The high level of diversity within the metagenome provide an almost inexhaustible
source of new enzymes (Cowan, 2000).
As the minimal requirements for gene expression are a promoter for transcription and a
ribosome binding site upstream of the translation initiation codon, the construction of
metagenomic libraries in ordinary cloning plasmids such as pUC19 or pSK+ has proved
successful. Small insert libraries (2-8 kb) are typically constructed in plasmids such as
pSK+, which contain strong inducible promoters such as lacI and T7 and/or T3 upstream
of the multiple cloning site (MCS) and support expression in cases where the insert does
not contain a promoter (Henne et al., 2000). Shotgun cloning is random and inserts of
varying sizes are cloned unidirectionally, which make systems with strong inducible
promoters on both DNA strands attractive prospects, thus ensuring transcription of genes
on both the positive and negative strands. Inducible promoters however, are not
82
essential as successful expression screening of a metagenomic library constructed in
pUC19 yielded 9 positive lipolytic clones (Ranjan et al., 2005).
Although small insert libraries have proved successful, large insert libraries (40-300 kb)
are preferred. In these systems, specialized plasmids such as cosmids, fosmids and BACs
which are able to maintain the integrity of large inserts, are used (Rondon et al 2000;
Lee et al., 2004). Large insert libraries are more informative, allowing access to
neighbouring genes or cis-elements required for effective expression of target genes,
which can easily be missed in small insert libraries. They are also more likely to provide
insight into the evolutionary origin of the functional gene using 16S rDNA analysis for
example (Streit and Schmitz, 2004). Expression screening using large insert libraries is
usually entirely reliant on native promoters due to the size of the inserts. The only
drawback is usually associated with the host cell not recognizing the heterologous
transcription signals. Another advantage of large insert libraries is the high level of
sequence coverage, which might allow for the reconstruction of whole novel genomes
(Venter et al., 2004).
Expression screening for the presence of lipolytic genes has been conducted in a number
of studies (Henne et al., 2000; Lee et al., 2004; Ranjan et al., 2005). Bacterial lipases
are typically extracellular enzymes, which enables activity screening on agar plates using
lipid substrates (Kouker and Jaeger, 1987; Feller et al., 1991; Henne et al., 2000).
Activity based screens for lipolytic enzymes involve nutrient media and a substrate such
as tributyrin for esterases or olive oil (trioleoglycerol) for lipases. The difference in
substrate selection is based upon the acyl chain length of the glycerolesters, where
83
longer chain lengths (>10) are only hydrolyzed by lipases and shorter chains (<10) are
hydrolyzed by both lipases and esterases. Tributyrin causes agar to become opaque and
hydrolysis of positive clones produces a distinct zone of clearance as lipolytic enzymes
hydrolyze the lipid substrate. Rhodamine dye-containing agar plates are pink. Bacterial
clones positive for lipase hydrolyze the trioleolglycerol substrate and the resulting uranylfatty acid ion forms a complex with the cationic rhodamine B, which is detected as an
orange fluorescence surrounding the colony under UV light (Kouker and Jaeger, 1987).
4.2 Aim
The principle aim of the work reported in this chapter was to detect lipase and esterase
genes using expression screening.
To achieve this, the following objectives were set forth:
)
Isolation of pure, intact high molecular weight DNA from MVG and PENP
)
Partial digestion using a tetranucleotide-specific enzyme
)
Construction of a PCR®-XL-TOPO® small insert metagenomic library from two of
the Dry Valley soils
)
Functional screening of the library for lipase activity
4.3 Results
4.3.1 Partial digestion and cloning of metagenomic DNA
High molecular weight DNA for tetranucleotide-specific digestion was obtained from soil
samples MVG and PENP using the modified Zhou protocol (Stach et al., 2001) as outlined
84
in Section 2.3.1. Routinely, the Zhou DNA extraction protocol yielded more DNA per
gram of soil compared to the Miller or BIO 101 DNA extraction methods. The DNA
isolated using the Zhou protocol showed little or no degradation, whereas both the Bio
101 and Miller protocols yielded less DNA which was more sheared. This prompted the
use of Zhou-extracted DNA for library construction.
1
2
3
4
14.0 kb
11.5 kb
Fig. 4.1
Metagenomic DNA from Dry Valley mineral soils. Lane 1, DNA molecular weight
marker; Lane 2, metagenomic DNA extracted using the Zhou protocol; Lane 3,
metagenomic DNA extracted using the Bio101 protocol; Lane 4, metagenomic DNA
extracted using the Miller protocol. The sizes of the molecular weight marker, phage
lambda DNA digested with PstI, are indicated to the left of the figure.
Trial digestions using three different tetranucleotide-specific enzymes (AfaI, AluI, HaeI)
was performed at time intervals varying between 40 and 60 min to determine optimal
restriction conditions. AluI yielded a smearing pattern ranging from intact high molecular
weight DNA to ~300 bp. However, smearing below 2 kb appeared less prominent, with
much of the partially digested DNA apparently between 2 kb and 10 kb (Fig. 4.2).
Following AluI digestion the endonuclease reactions were electrophoresed in a 1.0%
85
agarose gel. DNA ranging in size from 2 kb to 10 kb was excised and recovered using
GFX columns. The purified DNA fragments were A-tailed, dephosphorylated and finally
cloned into the PCR®-XL-TOPO® cloning vector as outlined in Chapter 2.
To verify the cloning, a small fraction (5 μl) of the total transformation reaction (1 ml)
was plated onto kanamycin selective media followed by incubation at 37°C overnight. The
remainder of the transformation mix was stored at 4°C until cloning efficiency could be
determined. Following overnight incubation the colonies were tallied and the theoretical
number of cfu.ml-1 was calculated. The library size was estimated at 90700 clones.
1
2
3
4
5
6
14 kb
1.7 kb
1kb/1.1kb
800 bp
Fig. 4.2
AluI restriction endonuclease digestion of MVG and PENP metagenomic DNA. Lane
1, DNA molecular weigh marker; Lanes 1, 2 and 3, MVG metagenomic DNA; Lanes
4 and 5, PENP metagenomic DNA. The sizes of the molecular weight marker, phage
lambda DNA digested with PstI, are indicated to the left of the figure.
86
4.3.2 Sequence space coverage
Assessment of the average insert size of the metagenomic library was performed using
restriction endonuclease digestion. Thirty colonies were randomly selected from agar
plates following library amplification and inoculated into LB-broth supplemented with the
appropriate antibiotic. Plasmid DNA was extracted as described in Section 2.15, followed
by restriction endonuclease reactions with EcoRI (Section 2.13). Insert sizes ranged from
1.7 kb to 5 kb with only two clones showing inserts at 0.8 kb (Fig. 4.3). Based on the
restriction analysis, the average insert size was determined at 3 kb and the library size
was calculated to contain 272 Mb of metagenomic DNA. Assuming the minimal number of
genomes (2000) (Sandaa et al., 1999) present in the mineral soil sample and an average
genome size of 4 Mbp, the library represented ~ 3.4% of the total metagenomic fraction.
1
4.5 kb
2
3
4
5
6
7
8
5.1 kb
9 10 11
5.1 kb
2.84 kb
2.0 kb
1.7 kb
1.1 kb
Fig. 4.3
Restriction analysis of PCR®-XL-TOPO® clones constructed by ligating partially
digested metagenomic DNA fragments. Lane 1, DNA molecular weight marker;
Lanes 2 through 10, recombinant PCR®-XL-TOPO® digested with EcoRI; Lane 11,
DNA molecular weight marker. The sizes of the molecular weight markers, phage
lambda digested with PstI, and EcoRI and HindIII are displayed to the left and right
respectively.
87
4.3.3 Library screening
4.3.3.1 Activity-based screening
To determine whether functional lipase or esterase genes were present in the
metagenomic library, expression screening of the cloned inserts was performed on 15 cm
indicator plates, prepared as described in Sections 2.2.5 and 2.2.6. Two copies of the
library were separately plated in aliquots of 60 µl (~2000 clones) onto esterase and
lipase indicator plates, and incubated at 37°C until colonies were visible. The plates were
then removed and incubated at 16°C for a period of 7 days. During the incubation the
plates were routinely monitored for the presence of clear zones surrounding the colonies
on esterase-specific plates and orange halos around colonies on the lipase-specific plates.
Following incubation for 7 days, no esterase of lipase activity was detected and
incubation was continued for another 5 days with no further change.
4.3.3.2 PCR-based screening of the metagenomic library
In order to probe the basis of the failure to detect functional lipolytic expression, PCR
analysis of the library was performed following the absence of both esterase and lipase
activity on indicator plates. A single copy of the complete library was subjected to
alkaline lysis (Section 2.16) to isolate the plasmid DNA for PCR analysis. PCR was
performed using degenerate primers LipF and LipR2 (Section 2.23). No PCR amplicons
were detected following PCR (Fig. 4.4).
4.4 Discussion
To construct an effective library, sufficient high molecular weight DNA needs to be
isolated from the environment or organism of interest. For this study, DNA was extracted
88
1
2
3
4
5
6
7
8
600 bp
400 bp
200 bp
Fig. 4.4
Agarose gel electrophoretic analysis following PCR of the PCR®-XL-TOPO®
metagenomic DNA library using primers LipF and LipR2. Lane 1, DNA molecular
weight marker; Lane 2 through 5, metagenomic library template; Lane 6, negative
control containing no template DNA; Lane 7, positive control using a recombinant
plasmid harbouring a full-length lipase gene of B. multivora; Lane 8, Negative
control containing only plasmid DNA. The sizes of the molecular weight marker,
phage lambda digested with PstI, are displayed to the left of the figure.
from oligotrophic soils using three different techniques, and the quality and yield
compared. Two methods, Miller and Bio 101, were mechanical extraction methods,
whereas the third (Zhou) was a chemical extraction. Analysis of extracted DNA following
agarose electrophoresis revealed that the chemical lysis produced intact high molecular
weight DNA consistent with other studies (Stach et al., 2001; Niemi et al., 2001). The
robust mechanical extraction methods yielded fragmented DNA with little or no high
molecular weight DNA. DNA extracted using the Zhou method was used in partial
restriction endonuclease digestion.
89
Library construction proved successful, as was evident from the average insert size (Fig
4.2), although the number of clones generated was lower than generated in other studies
(Henne et al., 2000; Majerník et al., 2001; Ranjan et al., 2005). No lipase activity was
detected following activity-based screening, which could be attributed to a number of
factors.
One such factor might be low nucleotide coverage of the metagenome, implying that the
fraction of DNA captured during library construction was not large enough to include a
functional lipase gene, although the average insert size was adequate to represent fulllength genes (3 kb). Statistically, libraries of 107 clones need to be screened to ensure a
positive hit (Gabor et al., 2004), whereas the metagenomic library only constituted
90700 clones (<104). For metagenomic libraries, the number of clones (Np) required to
recover a target gene at least once with the probability P can be derived from the
binomial distribution:
ln(1-P)
NP =
I–X
ln 1 -
G·c·z
Where I is the average insert size and X the size of the gene of interest. G represents the
average genome size present in the sample and z is the number of genomes assuming
even distribution (Gabor et al., 2004). The correction factor c reflects whether expression
of the insert is independent from the plasmid (relies on a native promoter and ribosome
binding site) or dependent on a plasmid-borne promoter and/or ribosome-binding site
(rbs).
90
The distribution of species (z) as given by the equation assumes that all species present
are
represented
equally.
This
assumption
however,
might
lead
to
a
gross
underestimation of the biodiversity within a given environment as shown by Gans and
co-workers (2005). By using reassociation kinetics two statistical models were proposed,
one assuming equal distribution and the second assuming uneven distribution of species.
Uneven distribution calculations indicated higher species abundance compared to even
distribution figures. Thus, if the calculated species estimates holds true for uneven
distribution, the value of z according to Gabor et al. (2004) should be much larger
implying an even larger theoretical Np.
Assuming the following for the Antarctic Dry Valley metagenomic library:
Average insert size (I) = 3 kb; Average lipase gene size (X) = 1.5 kb; Average genome
size (G) = 4 Mbp; c = 1; z = 2000; with a probability P of 0.9 of achieving a positive hit
during expression screening, the theoretical library size should constitute 1.2 × 107
clones, a figure substantially larger than the current library size. The estimated species
size (z) of 2000 is based on findings by Sandaa et al. (1999), where they indicating that
extreme metal contaminated soils contain approximately 2000 species. Even if the figure
of 2000 species for the Dry Valley soils is overestimated ten fold, then the number of
theoretical clones required for a 0.9 probability of obtaining a positive hit still far exceeds
the current size of the library.
Although the PCR®-XL-TOPO® vector harbours an inducible T7 promoter on the negative
strand, induced transcription was never performed. Instead, expression was dependent
on the native promoter and rbs. The use of independent gene expression allowed for
91
lower levels of gene expression as high levels of subfamilies I and II lipases have been
shown to be toxic to heterologous hosts (Bell et al., 2002). Additionally, successful gene
expression and isolation of lipases was obtained from small insert libraries (Ranjan et al.,
2005).
The metagenomic DNA used in constructing the library might not only be from bacterial
origin and could also represent DNA from both fungal and Archaeal taxa. Although the
presence of eukaryotes in the Antarctic mineral soils is limited to a few lichens, yeasts,
protozoa and bacteria (Friedmann, 1993; Ah Tow and Cowan, 2004), the probability of
having included eukaryotic DNA in the metagenomic library cannot conclusively be ruled
out. Contrary to other studies (Brambilla et al., 2001), Archaeal-specific PCR did not yield
any signals, which might imply that Archaea are either absent from the mineral soils
analysed or, alternatively, Archaea are present in levels below detection. The likely
presence of DNA sources other than that of bacteria could imply that less than 3.4% of
the metagenomic library represents prokaryotic DNA.
To verify whether the absence of lipolytic activity could be attributed to one of the factors
mentioned above, PCR analysis was performed on plasmid DNA extracted from an
amplified copy of the library. PCR has the advantage in that it does not require complex
regulatory mechanisms associated with lipolytic enzyme production, only the presence of
a nucleotide sequence. Using degenerate lipolytic primers in PCR analysis of the library
enabled the detection of a variety of lipolytic genes. Following PCR analysis of the
metagenomic library no signals were obtained in any of the PCR reactions and it was
concluded that no lipolytic genes were cloned from the environment.
92
To ensure sufficient coverage and the probable successful isolation of functional genes
using expression screening, the use of fosmids, cosmids or BACs should be considered.
These cloning systems allow for large inserts (>40 kb) and provide access to up- or
downstream genes or elements, such as chaperones, for functional secretion of target
enzymes. This especially holds true for family I subfamily I.1 and I.2 lipases (Arpigny
and Jaeger, 1999), as lipase specific foldases (lif) are essential for correct folding and
subsequent expression of its cognate lipase (Rosenau et al., 2004). Gene expression is
mediated by the native promoter and copy number is usually low (single copy per cell)
leading to low levels of enzyme, which is ideal, especially as strong inducible promoters
combined with high copy numbers could lead to stress on the host cell (Glick, 1995). In
addition, the net effect of large insert libraries further decrease Np due to larger inserts
and independent expression of target genes (Gabor et al., 2004).
93
Chapter 5
Prospecting for genes conferring lipolytic activity in
Antarctic mineral soils
5.1 Introduction
Using metagenomics to discover novel genes, pathways and bacteria instead of studying
pure isolated cultures from an environment, allows more efficient access to the available
information contained within the nucleic acid gene pool. Additionally, metagenomics
could allow for improved access to novel genes and sequence space.
The lack of sequence space coverage is clearly demonstrated by prokaryote community
analysis. During early culture-based techniques it was observed that very few of the
prokaryotes were cultured compared to the cells observed using other techniques such as
staining, microscopy and reassociation kinetics (Torsvik et al., 1990; Amann et al.,
1995). Molecular phylogenetic studies targeting the 16S rDNA gene greatly expanded the
then narrow window of available prokaryotes allowing both documentation of and access
to novel bacteria (Amann et al., 1995). Although molecular techniques continuously
expand sequence space, the accessible sequence space remains a fraction when weighed
against the total estimates of approximately 1030 individual microorganisms (Whitman et
al., 1998; Gans et al., 2005).
94
5.2 Aims
The primary aim of this chapter was to investigate whether degenerate primers, based
on known lipolytic gene sequences, would be able to detect putative cold-active lipase
genes within metagenomic DNA samples.
To successfully achieve this, the following techniques were used:
) The design of degenerate primers to specifically amplify gene fragments
harbouring α/β-hydrolase motifs from metagenomic DNA isolated from MVG and
PENP samples.
) Cloning and bioinformatic analysis of the gene fragments to ascertain possible
novelty.
5.3 Results
5.3.1 Degenerate primer design and PCR
5.3.1.1 Degenerate PCR using primer set OXF1 and ACR1
Probing for lipolytic genes within the Antarctic cold desert mineral soils, involved the use
of degenerate primers. Three different sets of degenerate primers were used to target
and PCR-amplify partial lipase gene fragments (Table 2.1). One of the primer sets used
(OXF1 and ACR1) were originally designed by Bell and co-workers (Bell et al., 2002) to
target lipase genes within a thermophilic environment. Primers OXF1 and ACR1 were
designed in silico using CODEHOP software (Rose et al., 1998). This resulted in a primer
pair with a 3’ degenerate core and a 5’ non-degenerate consensus clamp which would
95
allow higher annealing temperatures and better primer utilization. Initial PCR trials
yielded very faint bands following agarose gel electrophoresis (Section 2.22). Aliquots of
the PCR reactions were subjected to additional rounds of thermocycling to enhance the
PCR-signal, after which the PCR amplicons were cloned. Fourteen colonies displaying a
Gal- phenotype on IPTG/X-gal supplemented selective plates were selected and screened.
To verify whether all fourteen colonies were recombinant, the pGEM® – T easy plasmid
DNA was isolated and subjected to restriction endonuclease digestion using EcoRI. All
fourteen clones were recombinant, containing inserts varying in size between 340 bp and
400 bp (Fig. 5.1). Only one of the clones contained an insert harbouring putative α/βhydrolase characteristics
(Fig. 5.1, lane 3). The resultant recombinant clone was
designated Lip3. Further attempts to amplify partial fragments failed and it was
concluded that the 512-fold degeneracy of OXF1 and ACR1 might have been too high.
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15 16
800 bp
540-400bp
340 bp
Fig. 5.1
Restriction endonuclease digestion of recombinant pGEM® – T easy plasmids. Lane 1
and 13, DNA molecular weight markers; Lanes 2 to 12 and 14 to 16, recombinant
pGEM® – T easy plasmids digested with EcoRI. The sizes of the molecular weight
marker, lambda DNA digested with PstI, are displayed to the left of the figure.
96
5.3.1.2 Design and PCR using primer sets LipF and LipR and LipF and
LipR2
Based on the fact that primer sets OXF1 and ACR1 did not efficiently amplify additional
partial lipase gene fragments, a new set of degenerate primers, LipF and LipR, was
designed. Design of degenerate primers involved the amino acid alignment of various
true-lipase genes (Family I, subfamilies I-VI) (Arpigny and Jaeger, 1999) to identify the
most conserved regions. Regions showing the highest level of conservation were
identified as the nucleic acids spanning the oxyanion hole and active site of the lipolytic
enzymes (Fig 5.2). As the nucleotide sequence of both the oxyanion hole and active site
of all the true-lipases are not totally conserved (Table 5.1), both primers LipF and LipR
were designed to have a 64-fold degeneracy (Table 1.1) to allow access to a significant
portion of sequence space. Primers LipF and LipR were used to PCR amplify partial
lipolytic fragments from community DNA isolated from samples MVG and PENP. Although
agarose gel analysis revealed improved levels of amplification following PCR analysis with
primer set LipF and LipR, multiple PCR amplicons and significant background were
observed (Fig. 5.3). Following exhaustive PCR attempts it was determined that LipR was
not sufficiently specific, which might have lead to mispriming at the active site causing
multiple PCR amplicons. Primer LipR was redesigned to have a 32-fold degeneracy and
designated LipR2. Primer pair lip LipF and LipR2 yielded a single band of approximately
300 bp following PCR and gel electrophoresis (Fig. 5.4). The bands were recovered and
cloned into plasmid pTZ57R/T (Fermentas). Eight recombinant clones with were identified
based on blue/white selection and restriction analysis revealed fragments varying in
length from 240 bp to 300 bp (Fig. 5.5). To determine the origin of the cloned DNA
fragments, all
97
Fig. 5.2
Clustal W amino acid alignment of 18 true lipase sequences from family I, subfamilies
I and II as defined by Arpigny and Jaeger (1999). Similar amino acids are highlighted
in yellow and identical amino acids are highlighted in blue. The most conserved
regions, the oxyanion hole (aa 6 – 12) and active site (aa 113 – 118), are indicated
by arrows and served as the core regions for primer design.
98
Table 5.1
Conserved motifs of the oxyanion hole and active sites of true lipases from Family I,
subfamily I-VI.
Bacteria
Accession
number
Oxyanion hole
(Forward primer)
Active site
(Reverse primer)
P. aeruginosa
D50587
PIVLAHG
GHSHGG
P. fluorescence C9
AF031226
PLVLVPG
GHSQGS
V. cholerae
X16945
PIVLVHG
GHSHGG
P. fragi
X14033
PILLVHG
GHSQGA
P. wisconsinensis
U88907
PIVLVHG
GHSQGS
P. vulgaris
U33845
PIVLVHG
GHSQGP
B. glumae
X70354
PVILVHG
GHSQGG
B. cepacia
M58494
PIILVHG
GHSQGG
B. luteola
AF050153
PIILVHG
GHSQGG
B. subtilis
M74010
PVVMVHG
AHSMGG
B. pumilus
A34992
PVVMVHG
AHSMGG
U78785
PIVLLHG
AHSQGG
X95309
PIVLLHG
AHSQGG
M12715
PVVFVHG
GHSMGG
X02884
PFVFVHG
GHSMGG
AF090142
PVVFVHG
GHSMGG
X99255
PVILIPG
GHSQGG
U80063
PVVLNVG
GHSQGG
Subfamily I
Subfamily II
2 Subfamily IV
3 Subfamily V
B. stearothermophilus
B. thermocatenulatus
S. aureus
S. hyicus
S. epidermidis
4 Subfamily VI
P. acnes
S. cinnamoneus
99
1
2
3
4
5
6
800 bp
500 bp
340 bp
200 bp
Fig. 5.3
PCR amplification using metagenomic DNA from MVG and PENP as template. Lane
1, DNA molecular weight marker; Lanes 2 and 3, amplification following PCR using
MVG metagenomic DNA; Lanes 4 and 5, amplification following PCR using PENP
metagenomic DNA; Lane 6, Negative control containing no template DNA. The sizes
of the molecular weight marker, phage lambda digested with PstI, are indicated on
the left of the figure.
1
2
3
4
400bp
200 bp
Fig. 5.4
PCR amplification using MVG and PENP metagenomic DNA as template. Lane 1, DNA
molecular weight marker; Lane 2, MVG metagenomic DNA; Lane 3, PENP
metagenomic DNA; Lane 4, negative control containing no template DNA. The sizes
of the molecular weight marker, Hyperladder I (Bioline), are indicated on the left of
the figure.
100
1
2
3
4
5
6
7
8
9
10
11
340 bp
250 bp
200 bp
Fig. 5.5
Agarose electrophoretic gel of the recombinant pTZ57T/A cloning plasmid
containing different partial α/β-hydrolase gene fragments. Lane 1, DNA molecular
weight marker; Lane 2 – 11, recombinant pTZ57T/A digested with XbaI and BamHI.
The sizes of the molecular weight marker, phage lambda digested with PstI, are
indicated on the left of the figure.
eight clones were sequenced using M13 forward and reverse sequencing primers (Table
1.1)
5.4 Analysis of partial lipase sequences
In total, nine different partial DNA fragments associated with enzyme class 3.1.1 were
identified (Table 5.2 and 5.3). Nucleotide sequences were analysed for possible sequence
errors using BioEdit Sequence Alignment Editor version 5.0.2 and translated into all six
reading frames using the translate tool located at www.expasy.org. The correct reading
frame was identified by the oxyanion hole amino acids sequence N’- PIVLVHG – C’ and
extended for approximately 240 bp to the active site amino acids sequence N’ –
GHS(H/Q)GG – C’. Two of the identified partial lipase sequences were 300 bp in length.
After BLASTp analysis, all partial sequences showed varying degrees of similarity to
101
lipase genes from known bacteria and grouped as a variety of E.C.3.1.1 enzymes. Three
of the partial clones (240-1, 240-5, 300-5) were identified as being putative enzymes. All
but two of the partial fragments, 240-5 and 300-3, harboured conserved domains. The
two domains which featured most commonly amongst the gene fragments were MhpC
and PldB. MhpC is a functional prediction indicator and indicates that the amino acid
sequence belongs to the α/β-hydrolase superfamily. An enzyme containing an MhpC
domain is predicted to be either a hydrolase or acyltransferase.
Table 5.2
Clone
Partial lipase amplicons obtained following PCR of community DNA samples using
degenerate primer sets OXF1 and ACR1 and LipF and LipR2.
Nearest matches
Organism
Conserved domain
Identity
Alpha Beta
hydrolase fold
Sphingopyxis alaskensis
Abhydrolase 1, MhpC,
PldB
42%
240-1
Putative Esterase
Streptomyces coelicolor
MhpC
60%
240-2
Triacylglycerol lipase
LipA
39%
Lip3
Cryptococcus
neoformans
240-3
Hydrolase or
acyltransferase
Synechococcus elongatus
Thioesterase, pldB,
MhpC, COG3319, DAP2
35%
240-4
Class 2 lipase
Rubrobacter xylanophilus
Lipase 2, LipA, MhpC,
PldB
52%
240-5
Putative lipase
Streptomyces
cinnamonensis
None
39%
240-8
Hydrolase or
acyltransferase
Synechococcus elongatus
Thioesterase, MhpC,
LipA, PldB
50%
300-3
Esterase 2
Acetobacter pasteurianus
None
28%
300-5
Putative hydrolase
Symbiobacterium
thermophilum
DAP2, COG0412,
COG4188, PldB
61%
Identity implies the percentage identity the putative lipase gene has with that of the nearest match
The MhpC domain is also related to 13 other conserved domains. The PldB domain
suggests amino acid similarity to that of known lysophopholipases which are important in
102
Table 5.3
DNA and protein sequences of the partial lipolytic fragments isolated from the
Antarctic Dry Valley mineral soils
Clone
DNA sequence
Protein sequence
Lip3
CCTGTTGTCTTAGTTCACGGTGGTCCTGGAAGTCCATTGAGT
CCATATGCTGATGCTATATATGGTGAATGGGAAAGAGATTTTA
TTCTTGTTCAATGGGATCAAAGAGGGACAGGAAAAACTTATG
GACGTACTGCACCGGTTGAATTAAGCCCGGATTATTTAAAAT
CAAATCCATTGATTCTTGAACAGATGACCACAGATGGAATCG
AGCTTGCAGAATATCTTATTAAATACCTTGGAAAGCCAAAAAT
TATCCTTTTTGGAACTTCCTGGGGTTCAGTACCCGGGGTTCA
AATGGCTGTAAAACGCCCGGACCTTTTTTACGCTTATGTTGCA
CACTCCATGGGGGGCCTA
PVVLVHGGPGSPLSPYADAI
YGEWERDFILVQWDQRGT
GKTYGRTAPVELSPDYLKSN
PLILEQMTTDGIELAEYLIKY
LGKPKIILFGTSWGSVPGV
QMAVKRPDLFYAYVAHSMG
GL
240-1
GACCAATCGTCCTGGTGCACGGGGCGTGGCACGGCGGTTGG
TGCTGGAAGAAGGCGGTCCCGTTCCTGCGCGCGGCCGGACA
CGACGTCTTCACACCTACCCTGACCGGTCTCGGGGAACGCGT
GCACCTGCTCGCACCGGAGATCGATCTGACGACGCACGTCG
ACGACGTGCTCGGCGTGCTGGAGTACGAAGACCTCACCAAC
GTGGTCCTGGTCGGTCACAGTCACGGTGG
PIVLVHGAWHGGWCWKKA
VPFLRAAGHDVFTPTLTGLG
ERVHLLAPEIDLTTHDDVLG
VLEYEDLTNVVLVGHSHG
240-2
GACCGATTGTGCTCGTACATGGCTTGAGCGGCTTCAGTCGGC
TCATGCCGCGACGCAAGGCCATAAAGGGTTATTTCCCCGGCA
TCCAAGCGTATCTCGAAGCGAGTGGGAACCGGATCCTCTGCC
CGCGCGTCACGCCCACGGCGAGCGTGAGCACCCGCGCCCTC
GAACTGCGCACGACCCTCCTCCGCGAGTTCGGTTCGCAGCCC
TTCCATCTAATCGGCCACAGCCACGGTGGC
PIVLVHGLSGFSRLMPRRKA
IKGYFPGIQAYLEASGNRIL
CPRVTPTASVSTRALELRTT
LLREFGSQPFHLIGHSHGG
240-3
GACCGATCGTGCTGGTGCACGGCGCGCTGTTCACTAGCGCA
GGCTGGAGTCGCCTCCAGGGCGCGCTGCAGAGCCGCGGCTA
CAACGTCGTGACGCTCGACGTCCCAGGTCGCAACGGTGACG
GCCTCGATCCAAAGACCATCGACATCAACGACGCAGCGCAGA
AAGTTCGCGACGTCGTCGCTTTGCAACACGGTCCCGTGATTC
TCGTCGGTCACAGTCAGGGCGGC
PIVLVHGALFTSAGWSRLQ
GALQSRGYNVVTLDVPGRN
GDGLDPKTIDINDAAQKVR
DVVALQHGPVILVGHSQGG
240-4
GACCAATCGTCCTCGTGCACGGGACATTCGAGAACGCCAAG
CAGAACTGGGAGGTGCTCTCTGGTGAGTTGAAGACCAAGGG
GTACTGCGTCTTCGCGATCAACTACGGCACCAACGGGCTGAA
TCGCATCCAGAACTCCGCGAAGGGGTTGGACCGTTTCGTCGA
CAAGGTGCTGGAGTTCACGGGTGCCAAGAAGGTTCAGGTCG
TCGGCCATAGCCACGGCGGC
PIVLVHGTFENAKQNWEVL
SGELKTKGYCVFAINYGTN
GLNRIQNSAKGLDRFVDKV
LEFTGAKKVQVVGHSHGG
240-5
GACCGATTGTGCTGGTGCATGGCACCGGCGTCAGCCGCGAG
GAGAACTGGTCTTGGAACTACTGGAAGGTTCTCCCCACGGAG
GGCTTCGAGGTCTGCTGGGTGAAACTCCCCCGTGCGCCACTC
GGCGGCATCCAGATCGCATCCGAGTACGTAGCTCGGGCGAT
AGGTGTGATGCACCGTAGGAGCGGCGAACGGATCGACGTAC
TCGGCCATAGTCACGGCGGC
PIVLVHGTGVSREENWSW
NYWKVLPTEGFEVCWVKLP
RAPLGGIQIASEYVARAIGV
MHRRSGERIDVLGHSHGG
103
Table 5.2 continued
Clone
DNA sequence
Protein sequence
240-8
GACCGATCGTGCTGGTACACGGCGCGTTTCTGGGGAGCTGG
TCATGGAATAAGATCGTCAGTCGACTGCAGACCCAGGGGCAC
AATGCGATAATTCTCGACGTGCCCGGTCGAGCCGGAGACGC
TATCCTCCCGAGCGAAGTTACTTTGAAGTCGGCGGCTCAGAA
AGTCTGTACCGTTGCCGGTTTGCAGAAAGAGCCCGTGGTTKT
AGTAGGTCACAGCCAGGGCGGC
PIVLVHGAFLGSWSWNKIV
SRLQTQGHNAIILDVPGRA
GDAILPSEVTLKSAAQKVCT
VAGLQKEPVVLVGHSQGG
300-3
GACCAATTGTGCTGGTACACGGTTACAATTCAGACAACAGCG
CGTGGTTTCAGTCGGAAAGCTCGCCAAATCCAAACGATTTCC
TCGCGGCACTCCGAGAGGTCTTTCCGGCCGATTTCATTTTGC
CCGTGGAGTACGGCGTCGATCGCAGTGGCGCTGAACCTGAC
AACACGGAGAATACGTCGGGAGCGTTTGAGTACCTCGCTCC
GTTGCTCGATGCGCAACTGCGCACACTGGTAGAGAGTCCAAC
AAGCTTGTGGCATCAGCAGTGGGCCTTCACGCGCTACAACAT
TGTCGGCCACAGTCACGGTGGC
PIVLVHGYNSDNSAWFQSE
SSPNPNDFLAALREVFPADF
ILPVEYGVDRSGAEPDNTE
NTSGAFEYLAPLLDAQLRTL
VESPTSLWHQQWAFTRYNI
VGHSHGG
300-5
GACCAATTGTGCTGGTGCACGGGTCGGGACCGAACGACCGC
GACGAGACGCTCGGCGCGAACAAGCCCTTCCGCGATCTGGC
GCAGGGGCTGGCCTCGCAAGGCATTGCTGTCCTGCGTTACG
AGAAACGCTCGCGCCAGCATGCAGCCAAGACCGCCCAACAG
ACTAACTTGACCGTCAAGGACGAGACGATTGACGACGCGCTC
GCCGCCGTCCGTCTGCTCCGGCAAACTACTGTCATTGACGCG
AAAAAGATTTTCGTGCTCGGCCATAGTCACGGCGGC
PIVLVHGSGPNDRDETLGA
NKPFRDLAQGLASQGIAVL
RYEKRSRQHAAKTAQQTNL
TVKDETIDDALAAVRLLRQT
TVIDAKKIFVLGHSHGG
lipid metabolism. The LipA domain is indicative of predicted acyltransferase and
hydrolase functionalities.
5.5 Discussion
Obtaining novel biocatalysts traditionally involves either culture-based or expression
library screening. However, these two approaches do not allow access to the complete
available DNA complement provided by microorganisms leaving the available microbial
nucleic acid pool is greatly underused (Whitman et al., 1998). This is primarily due to the
fact that some 99% of the microorganisms appear to be uncultured (Amann et al.,
1995). Conversely, the use of metagenomic libraries holds promise of accessing totally
104
unknown sequence space, yet is compromised by the drawbacks of heterologous gene
expression (Lorenz, et al., 2002).
In this study, previously uncharacterized Antarctic mineral soils were probed for the
presence of genes that might confer lipolytic activity and broadly classify as α/βhydrolase enzymes. To achieve this, degenerate primers based on known lipase genes
were designed and the metagenomic DNA from the Antarctic mineral soils probed using
PCR. The partial DNA fragments were characterized using bioinformatic analysis.
The design and use of degenerate primers to target specific genes within environmental
DNA has been employed successfully (Eschenfeldt et al., 2001; Bell et al., 2002). Using
degenerate primers potentially allows access to all the target genes within the
community, although the efficacy is dependent on the level of degeneracy of the primers
and the known sequences used as template during primer design (Cowan et al., 2005).
The use of sequence-based screening is thought to be conservative because the
oligonucleotide primers used to identify target genes reflect conserved amino acid
sequences motifs (Lorenz et al., 2002). However, sequence-based screening as a means
to access sequence space might not be as conservative as predicted. Studies on the
adaptation and differences of psychro-, meso-, and thermophilic enzymes all indicate
that sites such as the active site remain highly conserved throughout all taxonomic
groupings
and
temperature
ranges
(Fields,
2001).
Rather,
substitutions
and
modifications which have led to enzyme enhancement have all been mapped to regions
and motifs located far from the active site as not to disrupt the interaction of the catalytic
residues (Fields, 2001). Additionally, studies on xylanases and polyketide synthases
105
demonstrated that novel enzyme sequence space could be retrieved using sequencebased approaches (Seow et al., 1997; Lorenz et al., 2002).
Combining sequence-based screening with community analysis might assist in obtaining
additional and even novel sequence space. 16S rDNA analysis of a community may
indicate both the community structure and which fraction of the community is
represented by uncultured microorganisms. If a very extreme environment is chosen the
diversity might be significantly less compared to a more temperate and less stressed
environment such as compost (Sandaa et al., 1999). Additionally, the gene to be
targeted will greatly influence the type of environment selected. If, for instance, a
protease gene is the target it might be more feasible to probe an environment high in
protein. This form of enrichment differs from that of laboratory enrichment in that
laboratory enrichment only selects for the culturable fraction within the sample because
artificial culturing is dependent on current techniques (Janssen et al., 2002).
The reason for obtaining only one partial lipolytic gene fragment using primer set OXF1
and ACR1 remains unclear. It was revealed that the limited conservedness of the lipase
genes restricted primer design and that PCR parameters had to be severely altered in an
attempt to optimize conditions (Bell et al., 2002 personal communication). In a separate
study (PT Basvi, unpublished) on Chinese hot spring sediment, it was found that the
OXF1 and ACR1 primers more readily amplified partial lipolytic fragments using published
parameters. Given that the OXF1 and ACR1 primers were originally aimed at targeting
lipolytic genes from a thermophilic environment, it might be concluded that this primer
set is better suited for thermophilic lipolytic enzymes.
106
Probing for lipase genes yielded nine partial lipolytic gene fragments (Table 5.2), which
was indicative of the presence of lipase genes within the Antarctic mineral soils. Although
bioinformatic analysis indicated identities varying between 35% - 61% with known
bacterial and fungal lipolytic genes, the true phylogenetic relationship of the fragments
remains unclear, as only partial gene sequences were known. This study however,
represents
the
first
where
cold-active
lipolytic
genes
were
targeted
from
an
environmental metagenomic sample using a PCR-based strategy. All previous studies
relied on cultured strains from environments such as Antarctica, Siberia and Alaska
(Table 1.2), which showed lipolytic activity when sub-cultured on indicator plates
(Arpigny et al., 1993; Choo et al., 1998; Suzuki et al., 2002).
Bioinformatic analysis of the partial lipolytic gene fragments indicated that they
originated
from
three
bacterial
phylotypes
(Actinobacteria,
Proteobacteria
and
Cyanobacteria) and one fungal phylotype (Basidiomycota). This is evidence that the LipF
and LipR2 primer set was capable of detecting lipolytic genes from both bacterial and
fungal origin. Of interest is that upon comparison of the partial gene fragments to
database entries none of the fragments showed even remote identity towards cold-active
lipases
identified
in
previous
studies,
including
those
from
Antarctic
bacteria
Psychrobacter immobilis B10, Moraxella strain TA144 and Psychrobacter sp. Ant300
(Feller et al., 1991; Arpigny et al., 1993; Kulakova et al., 2004). Whether this implies
that the putative lipase fragments are novel is unsure as they represent partial genes
and any attempts at concluding function and\or possible origin is speculative
107
A future strategy involving the partial lipase gene fragments would be to obtain the fulllength genes. This can be done by using the known sequence of the partial lipase gene
fragments as templates for the design of internal specific primers for each individual
gene. The up- and downstream flanking regions of the genes can then be obtained using
a variety of PCR-based techniques such as genome walking PCR (Kilstrup and
Kristiansen, 2000), TAIL-PCR (Liu and Whittier, 1995), subtractive hybridization (Felske,
2002) and inverse PCR (Hartl and Ochman, 1994). Although these techniques have
successfully been used to obtain flanking regions from single genome samples, their
efficacy on metagenomic samples is as yet undetermined.
108
Chapter 6
Summary and Conclusion
The study of microbial diversity is complicated by the fact that microorganisms are
microscopic and not easily differentiated morphologically (Schloss and Handelsman,
2004). Although current methods employed to assess microbial diversity remain
inadequate, the use of 16S rDNA analysis (Pace et al., 1986) and other fingerprinting
techniques such as DGGE and SSCP (Smalla, 2004) have allowed for increased access
and better understanding of microbial diversity. Efforts to fully determine microbial
diversity have led to the study of both extreme and diverse biotopes, which include sea
water, sub-seafloor, deserts, forest soil and snow to list but a few (Carpenter et al.,
2000; Dunbar et al., 2002; Webster et al., 2003; Venter et al., 2004). In this
investigation, 16S rDNA analysis was used to assess the microbial diversity of three
Antarctic mineral soils, each differing with respect to altitude, protein, lipid and water
content.
During the first part of the study, the community (metagenomic) DNA from all three
mineral soils (PENP, MVG and BIS) was probed using standard 16S rDNA primers E9F
(Farelly et al., 1995) and U1510R (Reysenbach and Pace, 1995). A 16S rDNA library of
each sample site was constructed followed by identification of unique clones using ARDRA
analysis. The findings indicated that the bacteria present within all three the sample sites
broadly grouped into eight phylogenetic groups, with Cyanobacteria, Actinobacteria and
109
Acidobacteria representing the majority of the phylotypes. A large fraction (21%) of the
sequences identified fall within the previously ‘uncultured’ class.
The current understanding of Antarctic mineral soil microbiology is based almost
exclusively on culture-based studies (Friedmann, 1993). However, a number of important
Antarctic Dry Valley microbial non-soil biotopes, including cryptoendolithic communities
(de la Torre et al., 2003), cryoconite holes (Christner et al., 2003), and lake ice and
marine ice flows (Priscu et al., 1998) have been subjected to detailed community
analyses using modern molecular phylogenetic techniques. This study represents the first
culture-independent phylogenetic analysis of Antarctic Dry Valley mineral soils. There is a
notable overlap between the phylotypes identified in the cryoconite hole and Dry Valleys,
which might be expected as wind-blown particules would colonise Dry Valley soils and
adjacent glaciers. Christner et al. (2003) reported the presence of Proteobacteria (α, β),
Actinobacteria,
Verricumicrobia,
Acidomicrobia
and
Cyanobacteria,
all
phylotypes
identified in the Dry Valley mineral soils. Cyanobacteria and Actinobacteria both
constitute major fractions of the phylotypes identified in both the Dry Valleys and
cryoconite hole, but the Acidobacteria are significantly more abundant in the Dry Valley
soils, with only a single clone identified in the cryoconite hole community. Other
phylotypes
unique
to
the
cryoconite
hole
were
Cytophagales,
Planctomyces,
Gemmimonas and γ-Proteobacteria. Within the Cyanobacteria Christner et al. (2003)
identified Chamaesiphon, Leptolyngbya and Phormidium sp. of which only Phormidium
was identified in the mineral soils. Priscu et al., (1998) focused on Cyanobacterial
communities in lake ice and found the same three species present as in the cryoconite
hole, which might not be surprising as cryoconite holes are formed in ice (Christner et
110
al., 2003). The cryptoendolithic study of de la Torre et al. (2003) identified several
phylotypes overlapping with the findings of this investigation. Cyanobacteria identified
within
the
cryptoendolithic
Chroococcidiopsis,
which
community
represent
include
Cyanobacteria
Gloeocapsa,
normally
Plectonema,
associated
with
cryptoendolithic communities (Nienow and Friedmann, 1993). It is evident that historical
data from culture-dependant studies do not accurately represent the true microbial
species diversity of the Dry Valley mineral soils and although this is a preliminary study
exhibiting small sample size over a narrow range, there is substantial motivation for
more elaborate investigations.
The presence of cyanobacteria within the PENP sample suggests further investigation, as
cyanobacterial populations are not usually associated with desiccated environments
(Miller and Bebout, 2004). In addition, no evidence of cyanobacterial mats were
observed near Lake Purgatory and the putative members identified within sample PENP
are not typically associated with cryptoendolithic communities, which might suggest the
presence of free-living cyanobacterial communities in the mineral soils. PENP is most
likely to contain evidence of an active community as cyanobacteria are capable of
photosynthesis at -5°C and nitrogenase activity has been shown to be stable at -7°C
(Pandey et al., 2004). Future work should include detailed surveys of cyanobacterial
distribution in relation to environmental and physical factors to understand the principles
which dictate the distribution of these soil-borne cyanobacterial populations.
The molecular analysis performed on the Antarctic mineral soils indicate a substantial
reservoir of microorganisms. Early studies relying on culture-based techniques only
111
identified a small fraction of microorganisms due to culturing constraints. This might
have led to the assumption that the mineral soils contain a restricted number of
cosmopolitan taxa (Cameron et al., 1972; Friedmann, 1993). Molecular phylogenetic
analysis allow more insight into community structures of various biotopes, identifying
both
known
and
unknown
or
‘uncultured’
microorganisms.
These
uncultured
microorganisms provide potential novel resources and could be used in various
metagenomic approaches to identify biotechnologically important enzymes or pathways.
A future investigation would be to determine which fraction of the phylotypes identified
form part of the extant population of microorganisms and to what degree they play a role
within the active community structure. DNA directly isolated from the mineral soils might
potentially yield phylotypic signals representing either metabolically active cells, VNBC
individuals or extracellular DNA (naked DNA) or a combination of all three. Although DNA
from dead cells released into the environment becomes available for degradation by
bacterial DNA nucleases, DNA-soil particle complexes shield naked DNA from digestion
for varying lengths of time (Recorbert et al., 1993). Survival of naked DNA in soils has
been shown to vary between 3 months, 23.5 weeks and 30 000 years for forest, Dry
Valley mineral soils and Siberian sediments, respectively (Stokstad, 2003; England et al.,
2004; Ah Tow and Cowan, 2005). Environmental factors such as the extreme cold, low
water availability and high levels of salts and desiccation may contribute to survival of
naked DNA within the Antarctic Dry Valley mineral soils. Before any conclusions can be
drawn, additional studies such as determining what proportion of the bacterial cells in the
soils is alive should be conducted. This can readily be ascertained by performing livedead staining followed by fluorescence microscopy (Savichtcheva et al., 2005). Designing
112
a study to determine if there is any naked DNA present in the soils should also be
conducted and the results can be compared to that of the live-dead staining. Thus, if a
large proportion of the cells in the soils are dead, the presence of naked DNA in the soils
should be expected. Conversely, if a large proportion of the cells are alive, studies
indicating bacterial activity using SIP (Pelz et al., 2001) and/or BrdU (Borneman, 1999)
should be performed.
If active cells are detected in the mineral soils additional studies should be performed to
determine which proportion of the phylotypes are dominant. To achieve this, sampling
areas surrounding the original site of collection and at various intervals throughout the
year will also yield valuable information regarding the distribution of the phylotypes.
Dominant and indigenous phylotypes to each mineral soil should be perpetual throughout
the year whereas exogenous phylotypes, for instance wind-blown individuals or
communities, would be periodic.
Another approach to study active cells might be to extract the total RNA complement and
to enrich for mRNA by subtractive hybridisation (Poretsky et al., 2005). Using random
primers, a cDNA population can be generated through RT-PCR. Genes such as 16S rRNA
could be targetted and. should be detected more readily as their copy numbers are
higher.
In the second part of the investigation a metagenomic library was constructed in the
PCR®-XL-TOPO® cloning vector and the library was screened for the presence of lipolytic
activity using expression screening in E. coli. Although a library of 104 clones was
113
generated no clones harbouring lipolytic activity were detected. Using statistical analysis
of the generated library it was concluded that the library was too small (i.e. represented
an estimated 3.4% of the metagenome) to represent even one copy of the desired gene
(Gabor et al., 2004). A possible way to increase the probability of obtaining a functional
gene during expression screening would be to increase the coverage of the metagenomic
DNA cloned. This could be achieved by either generating a library consisting of ≥107
clones, each containing an average insert size of 3 kb or by using systems which can
accommodate larger inserts such as fosmids, cosmids or BACs. The problem with
generating a library of ≥107 clones is that it is technically challenging to screen (Gabor et
al., 2004; Cowan et al., 2005), whereas using fosmids, cosmids or BACs greater
coverage can be achieved by generating a library 10-times smaller. Fosmids and related
systems allow the cloning of large (>40 kb) DNA fragments and provide access to up- or
downstream genes or elements, such as chaperones, for functional secretion of target
enzymes (Brady et al., 2001).
The third component of this study involved the PCR-based probing of a metagenomic
DNA sample for the presence of putative cold-active lipolytic genes. Degenerate primers
were designed to target the consensus DNA sequences of the oxyanion hole and active
site within the lipolytic genes. Initial PCR trials using primers OXF1 and ACR1 designed
by Bell et al. (2002) yielded only one partial gene fragment. The OXF1 and ACR1 primer
set appeared to work well for prospecting in thermophilic samples as Bell and co-workers
(2002) succeeded in isolating a single lipase gene from an olive-oil-enriched percolation
conducted at 65°C. Upon redesigning the primers eight partial putative lipolytic gene
fragments were identified. Bioinformatic analysis of the partial fragments revealed that
114
five of the sequences were predicted as either esterases or lipases and the remaining
three were more broadly classified as belonging to the hydrolase family.
This study indicated that the degenerate primers designed to probe for the presence of
lipolytic genes worked efficiently and it was concluded that the metagenomic DNA from
the Dry Valley mineral soils contained lipolytic genes. Although only partial genes were
isolated, bioinformatic analysis indicated that they possibly originated from three
bacterial and one fungal phylotype. The partial fragments also varied with respect to
known lipase genes and from one another. The extent of variation, novelty and possible
movement in sequence space remains speculative however, as none of the fragments
showed even remote identity towards cold-active lipases identified in previous studies.
Such relationships could only be ascertained once full-length genes had been acquired.
Future work could include extracting the full-length genes using either genome walking
or semi nested PCR. These two strategies have been used successfully in separate
studies (Bell et al., 2002; Eschenfeldt et al., 2001).
115
Portions of this work have been presented both nationally and internationally, and
published in peer-reviewed journals:
Congress Contributions
International:
Smith JJ, Ah Tow L, Cary C, Russell NJ, Cowan DA (2005) Phylogenetic analysis of
microbial diversity in Antarctic cold-desert biotopes. Synthesis of Soil Biodiversity and
Ecosystem Functioning in Victoria Land, Antarctica: An NSF Sponsored Workshop. April
2005: Jekyll Island, GA, USA.
Smith JJ, Ah Tow L, Cary C, Cowan DA (2004) Phylogenetic analysis of microbial
diversity in Antarctic cold-dessert biotopes. Extremophiles 2004: 5th International
Conference on Extremophiles, Cambridge, Maryland, September 2004.
National:
Smith JJ, Ah Tow L, Cary C, Cowan DA (2004) Phylogenetic analysis of microbial
diversity in Antarctic cold-dessert biotopes. 13th biennial Congress of South African
Committee for the International Union of Microbiological Societies SASM, Stellenbosch,
Cape Town, April 2004.
Smith JJ, Ah Tow L, Cowan DA (2003) Phylogenetic analysis of multigenomic Antarctic
samples. 18th congress of the South African Society of Biochemistry and Molecular
Biology SASBMB, Pretoria, July 2003.
Meyer Q, Smith JJ, Harrison STL, Burton SG, Soloman M, Cowan DA (2003). Recovering
novel genes from environmental DNA samples via a gene specific PCR method. 18th
congress of the South African Society of Biochemistry and Molecular Biology SASBMB,
Pretoria, July 2003.
116
Publications
Smith JJ, Ah-Tow L, Cary C, Cowan D (2005) Bacterial diversity in three different
Antarctic cold-desert mineral soils. Microbial Ecology 51:413-421.
Cowan, DA, Ah Tow, L, Smith, JJ, Cary, C, and Moodley, K. (2005) Microbial molecular
ecology of Antarctic mineral soil biotopes. In Antarctica; Global Laboratory for Scientific
and International Collaboration, A. Tan, Z Yasin, M Mansor, eds., Academy of Sciences
Malaysia, Kuala Lumpur, pp. 113-118.
Cowan DA, Arslanoglu A, Burton SG, Baker GC, Smith JJ, Meyer QC, Cameron R (2003).
Metagenomics,
gene
discovery
and
the
ideal
biocatalyst.
Biochemical
Society
Transactions, 32:298-302.
Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of Domain-specific 16S
primers. Journal of Microbial Methods, 55:5411-555.
117
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