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Low and High Nucleic Acid Content Bacteria: Reality or
Just a Flow Cytometric Phenomenon?
A Dissertation submitted in partial fulfillment for the degree of
Master of Science in Biotechnology
By
Sangeeta Jaiswal
School of Biotechnology,
KIIT University,
Bhubaneswar, Orissa
Under the supervision of
Prof. Dr. Thomas Egli
&
Dr. Frederik Hammes
Department of Environmental Microbiology
Swiss Federal Institute of Aquatic Science and Technology(EAWAG)
Dübendorf, Switzerland
1
Certificate
This is to certify that the thesis entitled “Low and high nucleic acid content bacteria: reality or just a
flow cytometric phenomenon?” submitted by Sangeeta Jaiswal pursuing master of science in
Biotechnology at School of Biotechnology, KIIT university, Bhubaneswar is an authentic record of the
work carried out by her under my guidance.
I recommend her project work to be submitted in partial fulfillment of the requirement for her
degree of Master of Science in Biotechnology of School of Biotechnology, KIIT University
Bhubaneswar, Orissa.
The assistance received by her from various sources during the course of study is duly
acknowledged.
Dr. Frederik Hammes
(Supervisor)
Department of Environmental Microbiology
Swiss Federal Institute of Aquatic Science and Technology
Dubendorf, Switzerland
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Acknowledgement
I would like to extend my thanks to Prof. Dr. Thomas Egli for giving me opportunity to carry out this
research at eawag and also for his great supervision. I owe my deepest gratitude to Dr. Frederik
Hammes for his patience, motivation, enthusiasm, and immense knowledge. His encouragement,
guidance and support from the initial to the final level enabled me to develop an understanding of
the subject.
I also express my gratitude to Dr. Mrutunjay Suar, Director of the School of Biotechnology, KIIT
University for providing an excellent environment for study & research and also for providing
invaluable opportunity for future. I would take this opportunity to thank Dr. Hans Peter Kohler for
his kind favor and support. Without him this thesis would not be possible.
My special thanks go to Prof. Peter Lüthy who has shown extreme interest and support towards my
master thesis and all other academic activities.
It is my pleasure to thank Dr. Chanakya Nath Kundu for his excellent guidance throughout my
master’s study. His inspirational advices have always helped me and will also help me to make my
future bright. I would like to thank all faculty members of School of Biotechnology who were always
supportive and helpful throughout my life at KSBT.
My thanks must also go to Karin for helping me in learning DGGE and ever pleasant and helpful
Stefan. I would like to thank all the people of Umik who have been very helpful during may project.
Further thanks go to Kiran, Simran and Lopa for cheering time we spent together.
Many thanks go to Rojee and Sarbari for the memorable time we spent together in hostel.
I should thank many individuals, friends and colleagues who have not been mentioned here
personally in making this educational process a success. May be I could not have made it without
their supports.
I offer my sincere gratitude to my parents for the efforts they have put for the perusal of my higher
studies and also my uncle who has supported me all through my life.
Lastly I thank to my sisters for encouraging me throughout my life.
Sangeeta Jaiswal
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Table of Contents:
Abbreviation…………………………………………………………………………………………………………….……………………….5
Summary…………………………………………………………………………………………………………………….................... ….6
1. Introduction………………………………………………………………………………………………………………………………..7
1.1 Background…….….…………………………………………………………………………………………………………………..7
1.2 Review of the literature………………………………………………………………………………………………………….8
1.3 Scope of the study………………………………………………………………………………………………………………..10
2. Experimental section…………………………………………………………………………………………….....................11
II
2.1. What limits a natural community into two clusters: stain accessibility or DNA
content? ……………………………………………………………………………………………………………………………...11
2.1. a. Introduction……………………………………………………………………………………………………………….11
2.1. b. Materials and methods………………………………………………………………………………………………11
2.1. c. Results……………………………………………………………………………………………………………………….12
2.1. d. Discussion………………………………………………………………………………………………………………….17
2.2. Isolation of low nucleic acid content bacteria from waste water and their growth
characterization by flow cytometry...........................................................................................19
2.2. a. Introduction…………………………………………………………………………………………………………………19
2.2. b. Materials and methods……………………………………………………………………………………………….19
2.2. c. Results…………………………………………………………………………………………………………………………20
2.2. d. Discussion……………………………………………………………………………………………………………………24
3. Conclusion………………………………………………………………………………………………………………………………..26
4. References………………………………………………………………………………………………………………………………..27
4
Abbreviations
Symbol
Abbreviation
µl
Microliter
AOC
Assimiliable organic carbon
ATP
Adinosine tri-phosphate
CTC
5-cyano-2,3-ditolyl tetrazolium chloride
DGGE
Denaturing gradient gel electrophoresis
DNA
Deoxyribonucleic acid
FCM
Flow cytometry
FISH
Fluorescence in situ hybridization
HNA
High Nucleic Acid
HPC
Heterotrophic plate count
LB
Luria bertani
LNA
Low Nucleic Acid
ml
Milliliter
ng
Nanogram
nm
Nanometer
PCR
Polymerase chain reaction
PCR
Polymerase chain reaction
PG
Pico green
RNA
Ribonucleic acid
rpm
Revolution per minute
SDS
Sodium dodecyl sulphate
SGI
SYBR Green I
SGII
SYBR Green II
SSC
Sideward scatter
SSCP
PCR single-strand conformation polymorphism
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Summary
The microbial community of any natural aquatic environment, when stained with nucleic acid stains
and analyzed by flow cytometry, tend to cluster into two distinct fractions when sideward scatter
(SSC) and green fluorescence is taken into account. One cluster shows low SSC as well as low green
fluorescence and the other one shows high SSC and fluorescence. Based on these observations these
two clusters have been given names LNA and HNA bacteria respectively. The majority of studies
carried out in this field have focused on the abundance of these HNA and LNA cells in different
environments and their relative activity. The main reason for this is a lack of suitable culture
methods. There are many conflicting results about the activity and phylogeny of these LNA and HNA
bacteria. HNA bacteria are generally regarded as active part of microbial community where as the
LNA are regarded as the inactive part. However few authors regard LNA as active bacteria. The aim
of this work was to better understand the underlying factors of this flow cytometry phenomenon.
Two major factors namely stain accessibility due to cell membrane permeability and DNA
compaction, were investigated for their influence on intensity of fluorescence. It was found that cell
membrane permeability does not play a significant role, but DNA compaction may play a major role
for low fluorescence intensity of LNA. Study of growth characteristics of an LNA isolate revealed that
fluorescence intensity and SSC is influenced by the growth phase of bacteria. The present study
pinpoints the limitation of classifying a natural community into low nucleic acid and high nucleic acid
content bacteria based on fluorescence intensity observed by flow cytomery. The fluorescence of a
bacterium may be influenced by many factors like cell membrane permeability, DNA topology, copy
number of chromosomal DNA, physiological state of bacteria etc., which was also reflected in the
study of growth characteristic of LNA isolate. So there exists a strong need to investigate the
multiple factors which are responsible for the clustering of natural community.
`
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1. Introduction
1.1 Background:
One of the most fascinating and attractive aspects of the microbial world is its extraordinary
diversity. Microbial diversity depends among other things on nutrients available in the environment
and on their varied concentrations (ranging from extremely low in the range of micrograms of
organic matter per litre to a level approaching those in laboratory culture media). Water provides an
excellent environment for a wide variety of microorganisms to survive and interact with each other.
These microorganisms also play an important role in the biogeochemical cycle. A major problem in
understanding these microbial activities and interactions is that most of the microorganisms cannot
be cultured on conventional rich media. Culture independent techniques like epifluorescence
microscopy, denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization
(FISH), etc. have been employed to overcome this problem (Boon et al., 2002; Muyzer et al., 1998).
But these methods are often time consuming and lack resolution and precision. Another technique
which is gaining popularity in microbiology is flow cytometry (FCM). Tens of thousands of cells can
be analyzed in a few minutes by FCM and multiparametric data generated by it is not only rapid but
also accurate. The ability of flow cytometer to distinguish particles on the basis of the fluorescence
makes it suitable for analysing cells on the basis of the amount of nucleic acid, respiratory enzymes
or other molecular markers (Lebaron et al., 2002; Longnecker et al., 2005b). FCM is emerging as a
powerful technique with wide range of application in microbiology. The following are the different
areas of microbiology in which FCM has been used.
Cell counting: Quantification of total bacterial population present in a sample is a basic and
essential step in several areas of microbiology including public health, food, drinking water, and
natural environments. Until recently, epifluorescence microscopy after fluorochrome staining was
used to obtain total count of bacteria in a sample. But this technique is laborious and time
consuming. Flow cytometry in combination with nucleic acid stains such as SYBR green I, SYBR green
II, SYTO green, Pico green has been proven to be a useful technique for counting all bacterial cells.
Planktonic bacteria are usually much smaller than cultured bacteria. They cannot be distinguished
from abiotic particles only on the basis of light scatter. Nucleic acid staining helps to separate abiotic
particles from cells. FCM has been used for microbial quantification in aquatic environmental
samples (Gasol et al., 2000). Cell counting is a routine process in drinking water treatment plants.
Conventional heterotrophic plate count (HPC) methods have many limitations as they rely on the
culturabilty of the microbial cells. Flow cytometry on the other hand counts each and every bacterial
cell present in water irrespective of its culturability and physiological state (Hammes et al., 2008).
Quantification of cellular DNA: The Presence of stains which bind specifically to DNA has made
determination of the DNA content of bacteria possible. Button and co-workers (2001) have shown
how the DNA content of aquatic bacteria can be determined with the help of flow cytometry. Even
the copy number of bacterial chromosome can be determined by FCM (Muller et al., 2007).
Determination of the cell size: Conventionally forward scatter is taken as indicator of particle
size, but the sensitivity of forward scatter for the measurement of changes in size of small particle is
not sufficient so sometimes sideward scatter (SCC) is considered as indicator of size (Servais et al.,
2003; Wang et al., 2009).
Cell viability analysis: Cultivation dependent viability assay is not able to determine the viability of
unculturable and viable but unculturable (VBNC) bacteria along with culturable bacteria. Flow
cytometry not only provides rapid assessment of all types of cells but also gives accurate counts
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(Berney et al., 2008; Falconi et al., 2008). Moreover it can also provide information about the
damage of cellular component if stains which bind to specific target site are used.
The use of FCM in aquatic microbiology has unveiled many facts about unexplored aquatic microbial
community. One of the most interesting areas of research that has emerged due to the use of FCM is
the classification of aquatic microbial community in two groups. FCM analysis coupled with nucleic
acid staining suggests that natural aquatic communities tend to cluster into two groups: one
showing low fluorescence and low side scatter while the other showing high fluorescence and SCC.
These two groups have been named variously by different authors for example “group I” and “group
II” cells by Li and co-workers (1995), “low-DNA (LDNA) bacteria and high-DNA (HDNA) bacteria” by
Gasol and co-workers (1999) and “low nucleic acid (LNA) bacteria” and high nucleic acid (HNA)
bacteria by Lebaron and co-workers (2001). This classification is based on the assumption that the
amount of fluorescence produced is directly related to the apparent cellular nucleic acid content of
the bacteria. However SSC is also regarded as the indicator of apparent cellular size which suggests
that the LNA are smaller in size whereas the HNA are larger in size. The small size increases surface
to volume ratio which facilitates the nutrient uptake in small bacteria in low nutrient environment.
Reduction in cellular size can be regarded as a survival strategy in low nutrient environment. But the
significance of presence of low nucleic acid content bacteria and high nucleic acid content bacteria in
environment is still unknown.
Fig. 1: FCM graph showing LNA cluster has low green fluorescence as well as low SSC and
HNA cluster has high green fluorescence intensity and SSC (Wang et al., 2010).
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1.2 Literature review:
The low and high nucleic acid content bacteria have been found to be present in marine (Zubkov et
al., 2001; Lebaron et al., 2002; Longnecker et al., 2005) as well as fresh water (Bouvier et al., 2007)
environment however the ratio of LNA to HNA cells are different in different environment. This
variability was explained on the basis of predation. Predators have usually preference for live and
large bacteria. It was proposed that as HNA cells are active, live and larger than LNA. So HNA
bacteria are more prone to predation than LNA bacteria. Thus increase in predation decreases the
abundance of HNA population and vice versa (Gasol et al., 1999). Most of the studies carried out in
this field have focused on the abundance of these two groups of organisms in different
environments. All attempts to describe this flow cyotomteric phenomenon in terms of activity and
phylogeny have resulted in contradiction.
Activity of LNA and HNA cells: Several authors have described the occurrence of LNA and HNA
clusters in different way. Gasol and co-workers (1995) found a relation between size and activity of
bacteria in aquatic environment and demonstrated on the basis of CTC (5-cyano-2,3-ditolyl
tetrazolium chloride) reduction that active cells tend to be larger than inactive cells. The basis of
differentiation of active cells from inactive cells was that active cells reduce CTC into insoluble
fluorescent formazan. In subsequent study they described low fluorescent cells as LDNA bacteria and
high fluorescent cells as HDNA bacteria and found that LDNA bacteria are smaller and less dense
while HDNA cells are larger and more dense (Gasol et al., 1999). On the basis of leucine
incorporation and cell sorting experiment it was found that HDNA cells are active and live while
LDNA cells are inactive and dead. They argued, “if the LDNA particles include active bacteria, why
should there be a clear separation between both (high and low fluorescence, HDNA and LDNA)
bacterial groups?” The LDNA bacterial cluster was proposed to contain at least three types of
particles: inactive cells, recently dead bacteria and fragments of cells that still have pieces of DNA
able to bind the stains. Inactive cells can be assumed to have less DNA than growing cells due to
their non replicative state or can lose extra DNA such as plasmid or copies of same gene. Lebaron
and co-workers (2001) supported the finding of Gasol and co-workers (1995). However they
renamed these two clusters as low nucleic acid and high nucleic acid content bacteria on the basis of
the fact that nucleic acid stains bind to both DNA and RNA (Lebaron et al., 2001a). HNA bacterial
group were proposed to contain intact cells with at least a single genome with wide range of
activities (Lebaron et al., 2001a). Again while determining the relationship of cell specific activity
with scatter and fluorescence signal it was found that cells with highest scatter signals have 10 fold
specific activity than lowest scatter signal. Same tendency was found to a lesser extend for
fluorescence (Lebaron et al., 2002). Based on these observations it was concluded that HNA
fractions are live and active but there exists heterogeneity in the activity of HNA cells. However in a
separate study Longnecker and co-workers (2005) found that LNA may show somewhat low cell
specific metabolic activity in oligotrophic systems. Most contradictory results were obtained when
Wang and co-workers (2009) isolated and cultivated LNA bacteria from three different environments
and showed that these isolates were able to maintain the typical “LNA fingerprint” during growth.
Phylogenetic relation of HNA and LNA clusters: Based on DGGE pattern Longnecker and coworkers (2005b) suggested that “cells, may be present in both the HNA and LNA assemblages at
same time and that their ability to switch between the two assemblages is not a function of their
phylogenetic diversity”. But an exception to this observation was the presence of different
phylotypes, more specifically Cytophaga- Flavobacterium cluster; within Bacterioidetes, only in HNA
fractions. This phenomenon was explained on the basis of the fact that Bacterioidetes are involved
in the breakdown of high- molecular weight organic matter and perhaps the metabolic process used
for the consumption of high molecular-weight organic matter require the organism to maintain
larger genomes or multiple copies of the genome within same cells. Phylogenetic analysis of HNA
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and LNA clusters with PCR single-strand conformation polymorphism (SSCP) technique also revealed
the presence of same group of organisms in LNA and HNA clusters. (Servais et al., 2003). Servais and
co-workers (2003) also found quantitative difference for a few populations within LNA and HNA
fractions and concluded that “in natural aquatic ecosystem, most bacterial populations, or at least
the dominant populations are divided into two categories of cells depending on their nucleic acid
content. This means that phylogenetically identical cells can be found in the natural environments in
very different physiological states, resulting in cells with very different apparent nucleic acid contents
and biovolume”. Bauvier and co-workers (2007) studied the cytometric characteristics of LNA and
HNA cells and proposed four possible scenarios: first scenario tells that LNA cells originate due to
inactivation or disintegration of HNA cells. Second scenario proposes the origin of HNA cells from
LNA cells due to DNA replication or presence of several copies of DNA. Third scenario hypothesises
that LNA and HNA are different organisms having their own characteristic size and nucleic acid
content. The last one suggests that both HNA and LNA clusters consist of distinct group of organisms
but they can change their state in either direction.
Fig. 2: Illustrated outline of the four potential scenarios that may be envisioned concerning the
nature of the HNA and LNA groups of bacterioplankton cells (Bouvier et al., 2007).
Thus studies done so far in this area are not sufficient to explain this FCM phenomenon. More
elaborate research is needed to explain the separation of a natural aquatic community into two
basic clusters based on SSC and fluorescence.
1.3 Scope of the study:
The ecological significance of the presence of LNA and HNA bacteria in almost all environments
remains unclear. The florescence and SSC of a cell may be affected by many factors such as stain
accessibility, DNA conformation and topology. Investigation about these factors in present study
revealed that stain accessibility may not play important role but DNA conformation and topology
may be one of the reasons for the lower fluorescence of LNA cells. And if the occurrence of LNA and
HNA cells in the environment is the manifestation of different physiological stage of the same
bacteria then one way to improve our understanding about this fluorescence and SSC distribution
would be to isolate and study the growth properties of the isolates. The present study demonstrates
that a bacterium may show change in fluorescence at different growth stages. Though this study
could not present a vivid picture of this fluorescence and SSC distribution, a few key questions could
be pinpointed, the detailed investigation of which can improve our understanding about this FCM
phenomenon.
10
2. Experimental section
2.1. What limits a natural community into two clusters: stain
accessibility or DNA content?
2.1. a. Introduction:
The bacterial cells present in a natural community are generally divided into two groups: LNA and
HNA bacteria based on FCM observation. The question is how far is this classification able to
describe the diversity of natural communities of aquatic environment? Can all microorganisms of a
natural environment be confined into two boundaries defined by sideward scatter and
fluorescence? Does the low fluorescence of LNA correspond to low amount of nucleic acid present
inside the cell or is due to the poor accessibility of stain to the nucleic acids of the cell? These are
questions still to be answered. The present study focuses on some of the above questions. Four
water samples from different environments were analyzed for the presence of LNA and the HNA
clusters and then effect of different stains and cell membrane permeability on the fluorescence of
LNA and HNA clusters were investigated. It was found that two clusters are consistently present in
all environmental samples tested and both clusters were clearly visible with different nucleic acid
stains and cell membrane impermeability cannot account for the occurrence of two clusters.
2.1. b. Materials and methods:
Sampling: Fresh water samples were collected in sterilized Schott bottles (1L) from four different
sources: river water from Chriesbach (Dubendorf, Switzerland), wastewater treatment plant effluent
(Dubendorf, Switzerland), tap water (Eawag, Dubendorf, Switzerland) and spring water.
Flow cytometry: Flow cytometric measurements were done as described by Hammes et al. (2008).
Water samples were stained with 10 µl/ml SYBR® Green I (1:100 dilution in dimethyl sulfoxide;
Molecular Probes, USA), and incubated in the dark for 10 minutes at room temperature before
measurement. If necessary samples were diluted just before measurement with 0.2µm filtered
mineral water. Flow cytometry was performed using CyFlow® Space instrument (Partec, Hamburg,
Germany) equipped with a 200 mW laser, emitting at a fixed wavelength of 488 nm and volumetric
counting hardware. Green fluorescence was collected at 488 nm, red fluorescence was collected at
630 nm and all data were processed with the Flowmax software ( Partec). LNA and HNA cell
populations were gated on two-parameter dot plot of green fluorescence (520 nm against sideward
scatter (SSC). The specific instrumental gain settings for these measurements were as follows: green
fluorescence = 520 nm, red fluorescence = 760nm and SSC=330 nm. The same setting and the same
digital gate were used for all experiments. Geometric mean (GMean) values for green fluorescence
and SSC for both the LNA and HNA cell population were recorded. Samples were measured in
triplicate. The standard instrumental error on FCM measurement was always below 3%. The
detection limit of the instrument used in this study was about 200 cell/ml (Hammes et al., 2008).
DNA isolation: Water samples were filtered through 0.45 µm (for HNA) and/or 0.22 µm (for LNA)
filters 47mm in diameter (Millipore Durapore membrane). The filters were placed into
microcentifuge tube and 537 µl of TE (pH 8) buffer and 30 µl lysozyme (5.67-5 g/ µl) were added
followed by incubation at 37oC on a shaker for one hour. In the next step 30 µl SDS and 3 µl
11
proteinase K were added and again incubated at same temperature and rpm for 30 min. Then 100 µl
NaCl (5M) and 80 µl CTAB was added and incubated at 65oC for 10 min. After cell lysis DNA was
extracted with 700 µl of chloroform: isoamyl alcohol (24:1) with subsequent centrifugation for 5
min. The Supernatant was carefully transferred into fresh microcentrifuge tube and 350 µl phenol:
chloroform and 350 µl phenol was added followed by centrifugation for 5 min. DNA was precipitated
with 0.6 volume of chilled isopropanol followed by centrifugation at 18oC for 15 min. All
centrifugation steps were performed at 13000 rpm. The DNA Pellet was washed with 70% ethanol
and then air dried and dissolved in sterile Millipore water.
2.1. c. Results:
Presence of LNA and HNA clusters in different environments:
Four water samples from different environments were analysed repeatedly by flow cytometry after
SYBR Green I staining for the presence of LNA and HNA clusters and it was found that two clusters
were consistently present in all water samples irrespective of date and time of sampling (fig. 3). The
mean green fluorescence and mean side scatter of both LNA and HNA cluster varied in different
samples but two clusters could easily be distinguished from each other and they always fall within a
boundary defined by mean SCC and mean green fluorescence. Mean fluorescence of LNA and HNA
clusters varied between 7-16 units and 70-120 units respectively while mean SSC varied between 3-5
units for LNA and 13-25 units for HNA cells (fig. 4).
SSC -
1000
SSC -
1000
A
B
100
SSC -
SSC -
100
10
10
R2
R2
R1
R1
1
1
1
10
100
1000
1
10
SSC -
1000
100
1000
FL1 -
FL1 -
SSC -
1000
C
D
100
SSC -
SSC -
100
10
10
R2
R2
R1
1
1
R1
10
100
1
1000
FL1 -
1
10
FL1 -
100
1000
Fig. 3: Flow cytometric analysis of water samples collected from different environments. A: river
water; B: wastewater; C: tap water and D: spring water. (Axes titles: FL1: green fluorescence,
520nm; SSC: side scatter, 330nm). Electronic gates R1 and R2 were used to separate LNA from
HNA; R1: LNA, R2: HNA.
12
Relative position of LNA and HNA cells on flow River water
cytometer
River water
Mean Side Scatter
1000
100
HNA
10
LNA
1
1
10
100
Mean Green Fluorescence
1000
River water
River water H
RB
River water
Waste water
River water
River water
River water
River water H
RB
River water
Waste water
River water
River water
River water
River water
spring water
Spring water
Fig. 4: Graph showing the relative position (geometric mean of SSC and green fluorescence) of LNA
and HNA cells of Different samples.
Comparison of three nucleic acid stains:
Different nucleic acids stains have different staining mechanisms and different quantum yield. The
intensity of fluorescence produced when they bind to their target molecule is also different. So next
step was to check whether different stains produce the same two clusters or not. For this two
different water samples (river water and wastewater) were stained with three different nucleic acid
stains namely SYBR Green I (SGI), SYBR Green II (SGII) and Pico green (PG). It was found that
clustering of the natural community is not affected by different stains. Two clusters could clearly be
discriminated but the cell count and the mean green fluorescence varied with stains. The total cell
count of both the water samples was always less when stained with PG. There is no significant
difference in total cell count in the case of SGI and SGII staining. Surprisingly in river water sample
SGII gives higher LNA count than SGI (fig. 5). The same result was also obtained with waste water
sample. Similarly for each water sample the mean green fluorescence varied among the three stains.
Mean green fluorescence of PG stained cells was always less than that of the SGI and SGII stained
cells. However the mean fluorescence of HNA population was more when stained with the SGI
whereas the mean fluorescence of LNA population was higher when stained with SGII (fig. 6). Fig. 7
shows that sideward scatter is not affected by different stains.
13
Cells /ml
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
HNA
LNA
PG
SG1
SG2
PG
RW
SG1
SG2
WW
Fig. 5: Changes in cell concentration of different water samples when stained with PG, SGI and
SGII. PG: Pico green, SG1: SYBR green I, SG2: SYBR green II, RW: river water and ww: waste water.
Error bar in the figure indicate the standard deviations of triplicate measurements.
120
Mean fluorescence (a.u.)
100
80
60
LNA
40
HNA
20
0
PG
SG1
SG2
RW
PG
SG1
SG2
WW
Fig. 6: Changes in the mean fluorescence of different water samples when stained with PG, SGI
and SGII. PG: Pico green, SG1: SYBR green I, SG2: SYBR green II, RW: river water and ww: waste
water. Error bar in the figure indicate the standard deviations of triplicate measurements.
14
30
Mean side scatter (a.u.)
25
20
15
LNA
10
HNA
5
0
PG
SG1
SG2
PG
RW
SG1
SG2
WW
Fig. 7: Effect of different nucleic acid stains on SSC of LNA and HNA populations. PG: Pico green,
SG1: SYBR green I, SG2: SYBR green II, RW: river water and ww: waste water. Error bar in the
figure indicate the standard deviations of triplicate measurements.
Effect of cell membrane permeability on SYBR Green I staining:
The intensity of fluorescence of nucleic acid stains depends upon the accessibility of the stain
molecule to the target molecule which in turn depends upon the cell membrane permeability. To
check whether the SGI molecules are equally accessible to LNA and HNA cells, the cells were treated
with an ATP analysis reagent which makes the plasma membrane permeable and then stained with
SGI. It was found that the cell count of LNA and HNA cells does not change with increasing
concentration of the ATP reagent (fig. 8). But fluorescence of LNA and HNA cells changed differently.
Fluorescence of LNA cells first increased somewhat and then gradually decreased with increasing
concentration of ATP reagent (fig. 9). The fluorescence of HNA cells did not show any increment
rather it decreased gradually with increased concentration of ATP reagent (fig. 10).
30000
25000
20000
LNA
15000
HNA
10000
Total
5000
0
SG1
SG1& 2ul SG1& 4ul SG1& 10ul
ATP reagent ATP reagent ATP reagent
Fig. 8: Effect of treatment with ATP reagent on cell count. SG1: SYBR green I, SG2: SYBR green II.
Error bar in the figure indicate the standard deviations of triplicate measurements.
15
Mean fluorescence
14
12
10
8
6
LNA
4
2
0
SG1
SG1& 2 ul SG1& 4ul SG1 & 10ul
ATP
ATP
ATP
reagent
reagent
reagent
Fig. 9: Effect of cell membrane permeabilization on the fluorescence of LNA bacteria. SG1: SYBR
green I, SG2: SYBR green II. Error bar in the figure indicate the standard deviations of triplicate
measurements.
120
Fluorescence
100
80
60
HNA
40
20
0
SG1
SG1& 2 ul
ATP
reagent
SG1& 4ul SG1 & 10ul
ATP
ATP
reagent
reagent
Fig. 10: Effect of cell membrane permeabilization on the fluorescence of HNA bacteria. SG1: SYBR
green I, SG2: SYBR green II. Error bar in the figure indicate the standard deviations of triplicate
measurements.
DNA content of LNA and HNA fractions:
Genomic DNA was isolated and quantified with Nano Drop, ND100 from LNA and HNA fractions in
order to find difference between DNA contents of LNA and HNA cells. River water which contained
LNA and HNA approximately in 1:1 ratio was filtered through 0.45 µm filter to separate HNA from
LNA. DNA was extracted from both fractions separately. In another experiment LNA and HNA
cultures were grown separately and then DNA was extracted from equal number of cells. The cell
number was monitored with the help of the flow cytometry. No major difference could be observed
in the DNA content of HNA and LNA fractions in both river water sample and cultured HNA and LNA.
The DNA ratio between HNA and LNA for river water sample was 1.25 and that for cultured cells was
1.17. These results indicate that LNA and HNA cells do not differ much at least in the genomic DNA
content.
16
Table1: Comparison of genomic DNA content of LNA and HNA bacteria
No of HNA and LNA cells
DNA isolated from HNA cells (ng)
DNA isolated from LNA cells (ng)
Ratio of HNA to LNA DNA (ng)
River water
1.3×106
550
690
1.25
Waste water
109
2790
2390
1.17
2.1. d. Discussion:
Presence of LNA and HNA population in different environments
Separation of the natural micro flora into two groups based on SSC and fluorescence is a general
FCM phenomenon. These two groups of organisms have been reported to be present in sea water (Li
et al., 1995; Gasol et al., 1999; Lebaron et al., 2001), brackish water (Lebaron et al., 2001) and fresh
water (Bauvier et al.2007). Our study also reveals the presence of two distinct populations in river
water, waste water, tap water and spring water. These LNA and HNA clusters were found to be
consistently present irrespective of time and source of sampling. Their SSC i.e. size and
fluorescence which is generally related to the nucleic acid content always fell within same range
which indicate that their relative position does not change with source environment (fig. 3). Natural
communities consist of diverse group of microorganisms which can possess a range of cellular sizes
and nucleic acid content. But the factors that divide the natural communities into two groups are
still not known. It has been observed that at least for natural community low fluorescence is always
associated with small size and vice versa. So the questions arise: is it actually the amount of nucleic
acid that is responsible for the occurrence of two clusters on flow cytometer or there exists some
other factors that segregate the fluorescence of natural communities. The occurrence of LNA and
HNA populations have been related to activity of bacterial community and it has been proposed that
the HNA clusters consists of live and active bacteria whereas the LNA bacteria are regarded as dead
and inactive( Gasol et al., 1999; Lebaron et al.,2001 and Servias et al., 2003). Some authors suggest
that LNA are also live and active (Longnecker et al. 2001). Wang and co-workers (2009) successfully
cultivated LNA bacteria and showed that the LNA bacteria actively grow, multiply and retain their
“LNA fingerprints” (that is low fluorescence intensity and low-SSC signal). So it is clear that natural
communities do not separate into two groups based only on activity. It has been found that an
attempt to isolate LNA bacteria from a water sample without 0.45 µm filtration and dilution always
results in growth of HNA bacteria. This indicates that HNA are also active. So isolation of LNA and
HNA bacteria from same environmental sample and comparative study of their morphology and
physiology would provide useful information about their ecological significance.
Comparison of nucleic acid stains:
The three stains tested here have different target specificity, staining mechanism and quantum
yields. This explains the different fluorescence intensity obtained with different stains. It should be
noted that the cell count also vary with the type of stains. Pico green gives lower cell count than
other the two stains. So choice of stain can affect the result. SGI has higher preference for double
stranded DNA whereas the SGII has preference for RNA molecule. Mean fluorescence of LNA cells
17
was higher when stained with SGII. It may indicate that LNA cells are active and contain more RNA
than HNA cells.
Effect of cell membrane permeability:
The present study reveals that the cell membrane permeability is not responsible for the occurrence
of two clusters on FCM. Treatment with the ATP analysis reagent which makes plasma membrane
permeable does not alter the position of the LNA and HNA populations as well as the cell counts. The
fluorescence of LNA cells increases first and then decreases. Increase in fluorescence of LNA cells
may be attributed to the increase in permeability of cell membrane due to the ATP analysis reagent
treatment. Live death staining with SYBR Green /PI revealed that 2µl ATP reagent is sufficient to kill
bacteria (data not shown). So it can be argued that higher concentrations of ATP reagent cause
damage to nucleic acid which results in the decrease in fluorescence. HNA cells did not show the
initial increase in fluorescence rather their fluorescence decreased gradually with increasing
concentration of ATP analysis reagent. This indicates that there must be some difference in cell
membrane composition of LNA and HNA bacteria which makes HNA cells more susceptible to ATP
analysis reagent.
DNA content of HNA and LNA fractions:
Despite of the classification of natural communities into high and low nucleic acid content bacteria
there is no information available about the actual differences in the nucleic acid content of these
two groups of bacteria. The separate isolation of DNA from same number of LNA and HNA cells and
its quantification with the help of the nanodrop method reveals that LNA contain lesser amount of
genomic DNA than HNA. But the difference is not as high as indicated by flow cytometry. The ratio of
HNA and LNA genomic DNA was 1.25 for river water and 1.17 for cultured cells. The Slightly higher
ratio for river water may be due to the fact that while filtering large volume of water sample for
separation of HNA and LNA populations, few LNA cells are also retained on the filter. Less
fluorescence of LNA can be attributed to its small size. Chromosomes of small cells are usually more
compacted to be accommodated into small space available. Due to compaction many binding sites
may not be available for the stain which results in the lower fluorescence. However more reliable
methods are required for the quantification of the DNA content of LNA and HNA bacteria. Cellular
DNA content of aquatic bacteria can be quantified with the help of FCM and DAPI staining as
described by Button and co-workers (2001). But determination of size of genome of the LNA and
HNA bacteria by pulse field gel electrophoresis would be more reliable data for the estimation of
DNA content of LNA and HNA bacteria.
18
2.2. Isolation of low nucleic acid content bacteria from waste water
and their growth characterization by flow cytometry.
2.2. a. Introduction: Most of the bacteria in natural environment grow under low nutrient
environment and they do not grow on conventional rich media. The unconventional method of
cultivation of LNA bacteria on natural assimilable organic carbon as described by Wang and coworkers (2009) has provided new approach to study LNA bacteria in detail. Isolation and cultivation
of more LNA bacteria from different environments would result in the better understanding of
activity, phylogeny and their significance in the environment.
Hence the main ojective of the present study was to isolate LNA bacteria from waste water where
majority of bacterial population fall into LNA category and to study its growth characteristic by flow
cytometry. The present study shows that fluorescence and SSC signal of LNA isolate changes at
different growth phases. Our study raises question on the relevance of the terms “LNA” and “HNA”
as fluorescence of a cell may be affected by multiple factors.
2.2. b. Materials and methods:
Preparation of assimilable organic carbon (AOC) free vials:
Sterile, carbon-free glassware was prepared as described previously (Hammes and Egli, 2005). In
short, all glass bottles and caps were washed with detergent and then rinsed thrice. These were then
submerged overnight in 0.2 N HCl and again rinsed thrice. The vials were subsequently heated in
muffle oven to 550oC for at least 6h (to remove all races of organics). The caps were incubated at
60oC for 1 hour in 10% sodium persulphate, followed by rinsing with deionised water and finally air
dried.
Sample collection: wastewater was sampled in sterile 1 litre Schott bottle from a waste water
treatment plant (Dubendorf, Switzerland) and kept at 4oC until processing. The same water sample
was used for LNA isolation and denaturing gradient gel electrophoresis.
Flow cytometry: Nucleic acid staining and flow cytometry was performed as described above
(section 2.1. b.).
Enrichment and isolation of LNA bacteria: The LNA population of wastewater were separated
from the HNA population by filtration through a 0.45 µm pore size sterile syringe filter. These LNA
cells were then serially diluted with autoclaved and filtered (0.2 µm) river water (Chriesbach,
Dubendorf, Switzerland) to the concentration of 100 cells per ml. It was used as an inoculum for the
first enrichment of LNA bacteria. River water (Chriesbcach, Dubendorf, Switzerland) was sampled in
1 L Schott bottle and autoclaved and then filtered through a 0.2 µm pore size sterile syringe filter
(PVDF, Millipore, USA) to remove particles. This water was used as a medium for LNA isolation. 1 ml
of the inoculum was inoculated into an AOC free vial containing 15 ml of media to obtain 100 cells
per vial concentration and incubated at 30oC for three days. After three days, cultures were analysed
by flow cytometry for the growth of LNA bacteria. The cultures showing growth of only LNA cells
were selected and further diluted to 10 cells per ml and used as inoculum. Ten ml of inocula with
concentration of 10 cells/ml was prepared and inoculated into 10 different vials in order to obtain 10
cells per vials cell concentration. Cultures were again incubated at 30oC for three days and analysed
by flow cytometer. The subcultures showing the growth of LNA were again subcultured to obtain
pure culture. This time the inoculum was 1 cell per vial.
19
Wastewater sample
Filtration through 0.45µm pore filter
Population of LNA bacteria
Serial dilution until 100 cells per ml
1ml inoculated into each vial (100 cells per vial)
Enrichment of LNA bacteria from 100 cells
Serial dilution until 10 cells per ml
1ml inoculated into each vial (10 cells per vial)
subculture of LNA bacteria from 10 cells
Serial dilution until 1 cell per ml
1ml inoculated into each vial (1 cell per vial)
Pure culture of LNA bacteria from one cell
Fig. 11: Schematic presentation of isolation of LNA bacteria from wastewater.
Growth characterization: A pure LNA isolate was inoculated into AOC free vial containing 30 ml
autoclaved and filtered (0.2µm) river water to the final concentration of 1000 cells per ml and
incubated at 30oC. One ml of sample was withdrawn every 2 hours and analysed by FCM. The
experiment was done in triplicate. The cell concentrations as recorded by flow cytometry were
plotted and the specific growth rate was determined.
Denaturing gradient gel electrophoresis (DGGE): DGGE was performed to check the purity of
the LNA isolate. DNA was extracted by CTAB method from the waste water (from which LNA bacteria
was isolated), HNA enrichment culture (from same waste water), LNA enrichment culture and LNA
pure culture. 16S ribosomal RNA was amplified with primer 341f and 518r. The amplification
program was as follows: initial denaturation at 94oC for 5 min, 30 cycles of denaturation at 950C for 1
min, annealing at 530C for 1 min, extension at 720C for 2 min and a single final extension at 950C for
10 min. The PCR product were loaded onto 8% polyacrlamide gels (V/V) having gradient of 45-60%.
The electrophoresis was run for 16h at 600C and 120V. Gels were stained with SYBR® Green I nucleic
acid gel stain for 20 min with gentle agitation. The single bands obtained on the gels were cut and
reamplified with same primers but without GC clamp. Genomic DNA of pure isolate was also
amplified with 63f and 518r primers. The PCR products were purified with QIAquick® PCR
purification Kit, QIAGEN Kit and sent for sequencing. Sequencing was performed by Mycrosynth
(Balgach, Switzerland). Sequence similarity search was performed with the BLAST programme of the
National
Centre
for
Biotechnological
Information
(NCBI)
http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).
20
2.2. c. Results:
Enrichment and isolation of LNA bacteria:
The fact that HNA cells not only show more fluorescence but also more SSC than LNA i.e. they are
larger in size than LNA cells, forms the basis of separation of HNA from LNA by 0.45um filters. It has
been found that most (but not all) of the HNA cells are retained on the filters when a water sample
containing HNA and LNA are passed through 0.45 um filter (Wang et al., 2007). In the present study
LNA populations for isolation of LNA bacteria were obtained by filtration (fig. 11 A & B). These cells
were then successfully cultured and sub cultured to obtain pure cultures. The LNA isolates were able
to grow on natural AOC at 30oC as well as 15oC. But they did not grow on diluted LB medium even at
dilution rate of 10,000. The LNA isolates showed the same fluorescence and side scatter as those of
the initial LNA population (fig. 11C)
SSC -
1000
SSC -
1000
A
B
SSC -
SSC -
SSC -
10
10
10
R2
1
R2
R2
R1
1
C
100
100
100
SSC -
1000
R1
R1
10
FL1 -
100
1000
1
1
1
10
100
FL1 -
1000
1
10
100
1000
FL1 -
Fig. 11: Flow cytometric picture of waste water before (A) and after 0.45 µm filtration (B) and
pure LNA isolate (C). (Axes titles: FL1: green fluorescence, 520nm; SSC: side scatter, 330nm).
Electronic gates R1 and R2 were used to separate LNA from HNA; R1: LNA, R2: HNA.
Growth Characteristics: The LNA isolate was grown on natural AOC in batch culture. Lag phase
could not be observed as LNA bacteria had already entered in the exponential phase when the first
flow cytometric measurement was done. They entered into the stationary phase after 40 hours of
incubation. The specific growth rate during exponential phase was 0.35 h-1 (fig. 12B). Growth curve
of the LNA isolate followed the normal pattern of lag, log and stationary phase (fig. 12A). But a new
pattern of change in SSC and fluorescence was observed (fig. 13 and 14). After 10 hours of
incubation when cells had entered into exponential phase the side scatter and fluorescence of the
bacteria increased significantly and their position on flow cytometer corresponded to the position of
HNA bacteria (fig. 15A). As LNA cells passed through exponential phase and reached stationary
phase they regained their typical LNA SSC and green fluorescence (fig. 15B). But in late stationary
phase their SSC and fluorescence again increased and did not change even after 7 days of incubation
(fig. 15C). But when these cultures were inoculated onto fresh medium their fluorescence and SSC
again decreased and they looked like LNA again.
21
natural logarithm of cells
per ml
A
Natural logarithm of cell
count
16
15
14
Replica 1
13
Replica2
12
Replica3
11
10
0
50
y = 0.347x + 7.353
R² = 0.996
16
15
Replica1
14
13
Replica2
12
Replica3
11
10
0
100
B
10
20
30
Time in hours
Time in hours
Fig. 12: Growth curve (A) and specific growth rate determination (B) of LNA isolate.
70
Mean Fluorescence (a.u.)
60
50
40
Mean
Fluorescence
30
20
10
0
0
50
100
Time in hours
Fig. 13: Change in mean green fluorescence of LNA isolate during different growth phases.
Side scatter (a.u.)
10
8
6
Side scatter
4
2
0
0
20
40
60
80
Time in hours
100
Fig14: Change in mean side scatter of LNA isolate during diffrerent growth phase.
22
SSC -
1000
SSC -
1000
A
B
100
C
100
SSC -
SSC -
SSC -
100
10
10
1
1
1
10
SSC -
1000
100
1000
10
1
10
100
1000
FL1 -
FL1 -
1
1
10
FL1 -
100
1000
Fig. 15: Flow cytometric picture of LNA isolate at different growth phase, A: 10h, B: 20h and C: 60h.
(Axes titles: FL1: green fluorescence, 520nm; SSC: side scatter, 330nm).
1000
10
12h
14h
16h
Mean Side Scatter
100
18h
HNA
20h
24h
10
87h
10h
37h
39h
44h
LNA
46h
60h
1
1
10
100
1000 87h
Mean Green Fluorescence
Fig. 16: Changes in Gmean fluorescence and SSC of LNA isolate during growth.
Denaturing gradient gel electrophoresis and 16S ribosomal DNA sequencing: Single bands
were obtained with all LNA isolates as well as LNA enrichment culture. It shows that the first
enrichment itself was pure. 16S ribosomal DNA sequencing and BLAST search revealed a close
similarity (99%) with Polynucleobacter cluster. DGGE of HNA enrichment indicates that LNA isolates
are different from HNA bacteria.
23
Fig. 17: DGGE of Waste water total community HNA and LNA enrichment culture and LNA isolates.
2.2. d. Discussion:
Isolation and Cultivation of LNA bacteria: Isolation and cultivation of LNA bacteria from waste
water again confirms the finding by Longnecker and co-workers (2005) and Wang and co-workers
(2009) that LNA are active and live opposite to the hypothesis put forward by Gasol and co-workers
(1999) and supported by Lebaron and co-workers (2001). Most of the natural bacteria do not grow
on conventional rich media as they require low nutrient condition for the growth. The LNA isolate
did not grow on 10,000 times diluted LB medium which shows that LNA isolate have preference to
certain carbon source. River water can be regarded as complex medium with various carbon sources
and low AOC concentration. Thus natural medium can be regarded as an excellent medium for
isolation of uncultivable bacteria.
Growth Characteristics: The most interesting finding of this study was a pattern in changes in
fluorescence and SCC during different growth stages which contradicts the results of Wang and coworkers (2009). Lebaron and co-workers (2002) also observed changes in fluorescence and SSC of
10 different isolates of marine environment. But these isolates were all HNA bacteria. The
fluorescence and SSC values varied greatly within species at given growth stages and between
growth stages of a given species.
The change in fluorescence at different growth stages: It can be attributed to: 1) change in DNA
conformation and 2) increases in amount of DNA and/or RNA inside cell due to accumulation of
multiple copies of DNA and higher activity respectively.
Chromosomal DNA inside a bacterial cell is usually organised into a nucleoid in which DNA remains
compact. In this state many stain binding sites may not be available which in turn may result in low
fluorescence. This DNA has to be unfolded during replication and transcription. In fast growing cells,
DNA replication and transcription occurs continuously which results in unfolding of DNA that makes
more stain binding sites available. Again in actively growing cells RNA content also increases as cell
synthesises new proteins required for cell division. This can explain the increase in fluorescence
during exponential phase but cannot explain the increase in fluorescence in the late stationary
phase. This hypothesis can be tested by measuring fluorescence intensity produced by a DNA
molecule when it is present inside and outside of the cell.
24
There are two conditions when a bacterium can accumulate multiple copies of DNA inside the cell.
One is when growth rates are too high and the other under starvation ( Lebaron et al.,1994; Throsen
et al.,1992).
Bacterial cell cycle can be divided into three phases
a. C phase: time required for DNA replication
b. D phase: time required for cell division
c. B phase: time between completion of cell division and initiation of DNA synthesis
If the growth rate is too fast, then the B phase gradually decreases and if the growth conditions are
even better then B phase disappears. The DNA synthesis continues even when the cell is dividing and
the new replication starts while the older replication is still running. This is called uncoupled DNA
replication which results in the accumulation of multiple copies of DNA inside the cells (Muller,
2007).
Another phase exists as proposed by Muller (2007) called pre-D phase. It is the phase between DNA
replication and cell division. If growth conditions are better than this phase disappears. However if
growth conditions are limiting then few bacteria have been found not to divide even after
completion of DNA replication. They stay in this phase (pre-D) until better growth conditions are
available. It has been found that cells enter into the pre-D phase after the exhaustion of carbon and
energy sources and if these cells are inoculated into fresh medium they start dividing immediately.
But this behavior have been proposed to be species specific and not substrate specific (Muller et al.
2000). Akerlund and co-workers (1995) also found accumulation of multiple copies of chromosome
in stationary phase cells. Accumulation of multiple copies of DNA under starvation condition has
been reported for many other bacteria (Lebaron et al., 1994; Thorsen et al., 1992).
The first increase in fluorescence when LNA isolate enters into the exponential phase may be due to
uncoupled DNA replication. As cells pass through exponential phase their growth rate decreases
with each cell receiving one copy of DNA. So mean fluorescence decreases. The increase in
fluorescence in the late stationary phase may be due to the accumulation of multiple copies of DNA.
The observation that fluorescence of LNA isolates decreases upon inoculation in fresh medium
indicates that an LNA can never become HNA as proposed by Bauvier and co-workers (2007); though
their fluorescence and SSC may change depending upon environmental and growth conditions.
Change in SSC at different growth stages: Side scatter of the LNA isolate followed the same pattern
as fluorescence. During growth, cells first grow in size and then divide. This accounts for the increase
in SSC signal during exponential phase. As cells approach the stationary phase their growth declines
but cell division continues resulting in reduction in cell size. The SSC signal decreased upon entry into
the stationary phase but it did not reach the initial value indicating that the bacteria grown under
laboratory conditions are usually larger than when present in environment.
In late stationary phase the SSC signal again increased. This increase in SSC is accompanied by the
increase in fluorescence. It has been found that cells with multiple copies of chromosome are usually
larger than cells containing single copy of genome (Akerlund et al., 1995, Lebaron et al., 1994). This
is probably to accommodate multiple copies of DNA inside the cell.
25
The question arises which bacteria is HNA bacteria? Bacteria with large genome size or bacteria with
small genome size but multiple copies of genomic DNA, because both will produce more
fluorescence. The presence of multiple copies of chromosome is special case which occurs under
unfavorable or starvation condition. Bacteria under this condition should not be regarded as HNA
bacteria. But FCM cannot distinguish between “true” HNA cells and cells with multiple copies of
DNA. This study raises question on the relevance of the terms “LNA” and “HNA” bacteria based on
FCM analysis. A bacterium should be called LNA bacteria only when it has small genome size and vice
versa. But there should be a threshold genome size above which a bacteria can be regarded as HNA
bacteria and below which a bacteria can be regarded as LNA bacteria. The next step in this research
field should be to define LNA and HNA bacteria in terms of genomic size and to investigate whether
the HNA cells present in natural samples are true HNA cells or cells having multiple copies of DNA or
both. This can be done by isolating more LNA and HNA bacteria from different environments and
determining their genomic sizes.
DGGE and sequencing: The 16S rDNA sequence matched 99% with the Polynucleobacter cluster.
In a previous study Wang and co-workers (2009) isolated LNA bacteria from three different
environments and all belonged to Polynucleobacter cluster. Organisms belonging to this group have
cosmopolitan distribution. They are not restricted to freshwater but are also observed in arctic,
temperate and tropical climates and sometimes contribute to total 50% of the total microbial
community (Hahn et al., 2003). All attempts to isolate an LNA bacterium have resulted in isolation of
Polynucleobacter. It may be attributed to either ease of isolation or their cosmopolitan presence. It
may be possible that the growth conditions provided in laboratory are more favorable or these
bacteria can acclimatize more easily to laboratory conditions than others.
26
3. Conclusion
The significance of the classification of natural communities based merely on fluorescence intensity
and side scatter as observed by FCM is still a matter of debate. Fluorescence intensity of nucleic acid
stain may be affected by many factors. Natural communities consist of diverse types of bacteria
which may vary in genome size and hence nucleic acid content. So in nature “true” LNA and HNA
may exist but flow cytometry alone cannot give the real picture because:
1. Cells infected with phages, containing multiple copies of plasmids and or genomic DNA and
with significant RNA content may fall into HNA clusters on FCM analysis. However these cells
should not be included in HNA clusters as these are specific cases.
2. Again inactive cells, recently dead cells, spores or fragments of cells which still have pieces of
DNA may fall into LNA cluster in FCM analysis.
These two reasons may account for the fact that few authors (Gasol et al., 1999, Lebaron et al.,
2001) find LNA as dead and inactive while others not (Longnecker et al., 2005; Wang et al., 2009).
Natural environmental conditions are not always favourable for all microorganisms present and the
fluorescence of cells is affected by growth conditions. In an environment both “true” low nucleic
acid content and high nucleic acid content bacteria or either of them may be present depending on
the environmental conditions and diversity of natural micro biota. Some environmental samples
even show one more than two clusters as in fig. 18 given below:
SSC -
1000
SSC -
1000
A
B
100
SSC -
SSC -
100
10
10
R2
R2
R1
R1
1
1
1
10
100
1000
FL1 -
1
10
100
1000
FL1 -
Fig. 18: flow cytometric picture showing one (A) and more than two clusters (B). (Axes titles: FL1:
green fluorescence, 520nm; SSC: side scatter, 330nm). Electronic gates R1 and R2 were used to
separate LNA from HNA; R1: LNA, R2: HNA.
This indicates that in nature bacteria which show fluorescence intermediate of that of LNA and HNA
or even more than HNA may exist. These two examples and results from growth study of LNA isolate
show that consistent occurrence of two clusters in almost all environmental samples is not just a
flow cytometric phenomenon. However it may or may not be related to DNA content as
fluorescence intensity is influenced by many factors. Even if it is related to DNA content, the terms
“Low” nucleic acid and “High” nucleic acid cannot draw the line between two clusters. So if at all
there is a need to classify bacteria in terms of their nucleic acid content it should be based on
genome size rather than on fluorescence.
27
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