Journal of Environmental Biotechnology
Vol. 12, No. 1, 33–38, 2012

Original paper (regular paper)

Expression of the gyrB gene as an indicator of
growth activity of Escherichia coli
Katsuji Tani2*, Takeshi Kobayashi1, Akiko Sakotani2, Takehiko Kenzaka2 and Masao Nasu1
1

Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University,
1–6 Yamada-oka, Suita, Osaka 565–0871, Japan
2
Environmental Science and Microbiology, Faculty of Pharmacy, Osaka Ohtani University,
3–11–1 Nishikiori-kita, Tondabayashi, Osaka 584–8540, Japan* Tel & Fax: +81–721–9742* E-mail: tanika@osaka-ohtani.ac.jp(Received; 16 November, 2011/Accepted; 7 May, 2012)

To determine whether the DNA gyrase gene (gyrB) can be used as an indicator of bacterial growth activity, its expression
levels were estimated in Escherichia coli under diﬀerent growth conditions. E. coli was cultured in liquid medium at various
temperatures, and its nucleic acid was extracted and purified. The copy numbers of gyrB mRNA in the log, log-stationary,and stationary phase cultures in Luria-Bertani broth at 37°C were 4.7, 0.015, and <10–6 (below the detection limit), respectively. When the generation times of E. coli were 0.42, 1.5, 5.3, and 9.9 h, the respective ratios of gyrB mRNA to DNA were
4.7, 1.5, 0.15, and 0.09. gyrB mRNA was expressed at high levels in growing E. coli cells. Nucleic acids were extracted fromsurface river water and river sediment samples. The ratio of gyrB mRNA to DNA for the E. coli-Shigella group was 1.3 inthe river sediment sample and 0.008 in the surface river water sample. These results suggest that gyrB mRNA expression
can be used as an indicator of bacterial growth activity and that the river sediment is a potential natural reservoir for the E.
coli-Shigella group.
Key words: gyrB gene expression, growth activity, Escherichia coli, river water

1. Introduction
Physiologically active bacteria are only a part of thediverse bacterial populations present in natural environments1,3,16,27). Determining the bacterial growth activity innatural environments is important for understanding bacterial habitats, survival, and growth conditions. Respiration,
enzymatic activity, and incorporation of specific substances
have been used as indicators of bacterial physiology or growth
activity12,19,22,25,29). The rate of incorporation of thymidine,
leucine, or their analogs is widely used for the measurementof bacterial growth rate in environments7,8). However, thesemethods have some limitations when determining the growth
activity of a specific bacterial species in a natural environment, because samples need to be incubated, thus causingchanges in bacterial activity. gyrB is a housekeeping genethat encodes the B subunit of bacterial DNA gyrase, whichplays an essential role in DNA replication. Regulation of
gyrB transcription is associated with the cell cycle in eachcell18), and hence, gyrB mRNA expression may be used as anindicator of bacterial growth. To investigate the relationshipbetween bacterial growth and gyrB mRNA expression, wequantified the copy number of gyrB mRNA in Escherichia
coli at each growth phase using real-time reverse transcription (RT)-PCR. Furthermore, we evaluated the utility ofmeasuring gyrB gene expression by comparing it with theconventional direct viable count (DVC) method14).
Consumption of water contaminated by human or animal
feces is an important cause of intestinal and systemic illnesses.
Estimates of the total coliform count are routinely used tomonitor fecal contamination in water, but the US Environmental Protection Agency has stated that E. coli counts
provide a better indicator of water quality28). Furthermore,a previous study showed that amount of this bacterium canbe used as a major indicator of fecal contamination in theriver environment2), while other reports have suggested that
E. coli can grow in soil6,24). However, studies have shownthat E. coli is non-culturable under adverse conditions, suchas low nutrient availability and/or low temperature20,21). The
present study investigated gyrB expression in E. coli to better
understand the habitat of this bacterium in natural riverenvironments.
2. Materials and Methods
2.1. Bacterial strains and growth conditions
Experiments were performed using E. coli K-12 W3110,
Shigella sonnei IID 969, Salmonella enteritidis IID 604,
Salmonella enterica serovar Choleraesuis IID1682, andVibrio vulnificus ATCC 27562. E. coli was cultured in Luria-

34

Tani et al.

Bertani (LB) broth (0.5% NaCl, 1% tryptone, 0.5% yeast
extract) at 37°C or M9 medium (0.5% glucose, 100 μM
CaCl2, 200 mM MgSO4, 1.36% Na2HPO4, 0.6% KH2PO4,
0.1% NaCl, and 0.2% NH4Cl) at 20, 22, or 25°C. Shi.
sonnei, Sal. enteritidis, and Sal. enterica serovar Choleraesuis were cultured in LB broth at 37°C. V. vulnificus wascultured in LB broth (3% NaCl) at 37°C.
2.2. Simultaneous recovery of RNA and DNA
RNA and DNA were recovered from E. coli culturesusing the Qiagen RNA/DNA System Midi Kit (Qiagen.
K. K., Japan) according to the manufacturer’s instructions.
RNA and DNA were simultaneously recovered from thesurface river water and river sediment samples using themethod described by Hurt et al.10) with modified concentrations of sodium dodecyl sulfate and cetyltrimethylammonium
bromide in the extraction buﬀer23). The extracted DNA andRNA were dissolved in 100 μL of TE buﬀer (pH 8.0) and80 μL of Tris-HCl (50 mM, pH 7.5), respectively. Twenty
units of DNase I (Roche Diagnostics, Indianapolis, IN),
10 μL of 60 mM MgCl2, and 10 μL of 20 mM CaCl2 wereadded to the RNA sample, and the mixture was incubatedfor 30 min at 37°C. After incubation, RNA was precipitatedwith isopropanol and resuspended in 100 μL of TE buﬀer(pH 7.5). Recovered RNA and DNA samples were stored at–80°C and –20°C, respectively.
2.3. Preparation of standard DNA and RNA
E. coli was cultured in LB broth at 37°C. Stationary
phase cultures were sampled and nucleic acids were extracted
and purified as described by Tsai and Olson26). In brief,
nucleic acids were incubated with 100 μg mL–1 ribonuclease
A (Sigma-Aldrich Co., St. Louis, MO) for 30 min at 37°C
to digest RNA, then purified using Tris-EDTA-buﬀered
phenol-chloroform-isoamyl alcohol (25 : 24 : 1, pH 8.0)
and ethanol precipitation as described by Iwamoto et al.11),
and dissolved in TE buﬀer [10 mM Tris-HCl (pH 8.0) and1 mM EDTA]. Extracted DNA was analyzed by electrophoresis (100 V, 30 min) on ethidium bromide-stained agarosegels (1%, wt/vol) to confirm the purity of chromosomal
DNA, i.e., without any fragmentation or contamination with
RNA. DNA concentration was determined by PicoGreen(Molecular Probes Inc., Eugene, OR) DNA staining, anda standard curve was constructed using a series of lambda
phage DNA (Takara Bio Inc., Japan) solutions (serial dilutions, 10–1,000 ng mL–1) in TE buﬀer. The lambda phage
DNA and bacterial DNA samples were diluted 100- or
1,000-fold with TE buﬀer and stained with 1/400 volumeof PicoGreen in a 96-well microplate. Fluorescence intensity(emission at 530 nm) was measured using a fluorescencemulti-well microplate reader (CytoFluor II; PreSpetive
Biosystems Inc., Framingham, MA) at an excitation wavelength of 485 nm. The copy number of genomic DNA wascalculated using the measured DNA concentration and themolecular weight of each E. coli W3110 chromosomal DNA(5.0×10–18 g molecule–1).

A plasmid (pGEM T-Easy Vector; Promega Co., Madison,
WI) containing the gyrB insert cloned from E. coli W3110 was
constructed.The gyrB insert was amplified by PCR with theprimers ESgyrB171F and ESgyrB448R (Table 1). The plasmid
DNA was linearized using NdeI (Molecular Biosystems Inc.,San Diego, CA). In vitro transcription was performed using
SP6/T7 transcription kit (Roche Diagnostics, Indianapolis,
IN). To eliminate DNA contamination, RNA was digested
with DNase I (Roche Diagnostics, Indianapolis, IN).
Measurement of RNA concentration was performed using
the RiboGreen RNA quantification kit (Molecular Probes Inc.,
Eugene, OR). The standard curve was generated with a seriesof 16S rRNA solutions (serial dilutions, 10–1,000 ng mL–1)
using the 16S rRNA standard solution included in the kit.
16S rRNA and in vitro transcripts of E. coli W3110 gyrB
clone were stained with 1/400 volume of RiboGreen ina 96-well microplate. The fluorescence intensity (emission
at 530 nm) was measured using a fluorescence multi-well
microplate reader (CytoFluor II; PreSpetive Biosystems Inc.,
Framingham, MA) at an excitation wavelength of 485 nm.
2.4. Real-time PCR and real-time RT-PCR of E. coli gyrBDNA and mRNA
The LightCycler system (Roche Diagnostics, Indianapolis,
IN) was used for real-time PCR and real-time reverse transcription (RT)-PCR of E. coli gyrB DNA and mRNA. To
quantify the gyrB DNA from E. coli, LightCycler DNA
Master Hybridization Probes (Roche Diagnostics, Indianapolis, IN) were used with 5 mM Mg2+, 0.5 μM of each primer(ESgyrB171F and ESgyrB448R), and 0.2 μM of each FRET
probe [ESgyrB376 (3'-FITC) and ESgyrB404 (5'-LightCyclerRed640)] (Table 1). After an initial denaturation at 95°C for
10 min, 50 PCR cycles at 95°C for 15 s, with annealing at 66°C
for 5 s and an extension at 72°C for 25 s, were conducted.
A standard DNA template of 5 to 5×106 copies per reactionof E. coli W3110 genomic DNA was used to construct a
standard curve for real-time PCR quantification. To quantify
gyrB mRNA of E. coli, LightCycler FastStart RNA MasterHybridization Probes (Roche Diagnostics, Indianapolis, IN)
were used with 3.25 mM Mn2+, primers (ESgyrB171F and
ESgyrB448R), and FRET probes [ESgyrB376 (3’-FITC)
and ESgyrB404 (5’-LightCycler-LCRed640)]. After reverse
transcription for 15 min at 61°C for 15 min, 50 PCR denaturation cycles at 95°C for 1 s, with annealing at 64°C for
10 s and an extension at 72°C for 15 s, were conducted. A
standard RNA template of 5 to 5×107 copies per reactionof in vitro transcripts of the E. coli W3110 gyrB clone wasused to construct a standard curve for real-time RT-PCR
quantification.
LightCycler data were analyzed using LightCycler software version 3.5 (Roche Diagnostics, Indianapolis, IN). The
concentrations and crossing points were calculated, which
represented the threshold cycle numbers where the fluorescence signal first exceeded the level of the background noise.
These were obtained using the second derivative maximum
function of LightCycler software, together with propor-

Gene expression as an indicator of growth activity

35

Table 1. Primers and probes for real-time PCR
Primer and probe

Target gene

Target position

Sequence

ESgyrB171F (forward primer)
ESgyrB448R (reverse primer)
ESgyrB376a (probe 1 for FRETc)
ESgyrB404b (probe 2 for FRET)
pgL1908f (forward primer)
Luc175r (reverse primer)
n-LucHP1a (probe 1 for FRET)
n-LucHP2b (probe 2 for FRET)

gyrB gene of E. coli
gyrB gene of E. coli

171–194
427–448d
376–401d
404–426d
1908–1927e
2063–2082e
2008–2029e
2030–2058e5’-aga aat tat cgt cac cat tca cgc-3’
5’-gta cac cgt gtt cgt aga tct g-3’
5’-ctg tcg caa aaa ctg gag ctg gtt at-3’
5’-agc gcg agg gta aaa ttc acc gt-3’
5’-agg aag ctt tcc atg gaa ga-3’
5’-cag cgt aag tga tgt cca cc-3’5’-tga aga gat acg ccc tgg ttc c-3’5’-gga aca att gct ttt aca gat gca cat a-3’

gyrB gene of E. coli
gyrB gene of E. coli
Luciferase gene
Luciferase gene
Luciferase gene
Luciferase gened

3’ FITC labeled
5’ LCRed640 labeled
Fluorescence resonance energy transferdNumbering of bases is E. coli numberingeNumbering bases of pGeneGRIPTM-Lucabctional and arithmetic baseline adjustments that were madeaccording to the manufacturer’s instructions. The standard
curve was generated by linear regression of the crossing
point versus the logarithms of the concentrations for eachstandard sample.
2.5. Recovery rate of DNA and RNA
To determine the DNA and RNA recovery rates, internal
standards of DNA and RNA were added to each sample andthe internal standards were quantified by real-time PCR andreal-time RT-PCR, respectively, after recovery. The luciferase
gene was used as the internal standard because it was absent
from samples used in the present study. A 1.7-kb PCR
amplicon of the luciferase gene was used as the internal DNA
standard and a 1.8-kb in vitro transcript of the luciferase
gene clone was used as the internal RNA standard. Realtime PCR and real-time RT-PCR of the luciferase geneand in vitro transcripts used pgL1908f (forward primer),
Luc175r (reverse primer), n-LucHP1 (probe 1 for FRET),and n-LucHP2 (probe 2 for FRET) (Table 1). The luciferase
gene was quantified using LightCycler DNA Master Hybridization Probes (Roche Diagnostics, Indianapolis, IN) with
5 mM Mg2+, 0.5 μM of each primer, and 0.2 μM of each
FRET probe. After an initial denaturation at 95°C for
10 min, 40 PCR denaturation cycles at 95°C for 15 min,with annealing at 60°C for 10 s and an extension at 72°C
for 15 s, were conducted. Luciferase transcripts were quantified using LightCycler-FastStart RNA Master Hybridization
Probes (Roche Diagnostics, Indianapolis, IN) with 3.25 mM
Mn2+. After reverse transcription at 61°C for 20 min, 40 PCR
denaturation cycles at 95°C for 1 s, with annealing at 60°C
for 10 s and an extension at 72°C for 15 s, were conducted.
2.6. Sampling of E. coli culture
The relationship between the copy number of gyrB mRNA
(gyrB mRNA expression) and the growth rate was determined using E. coli W3110 cultured in LB broth at 37°C,
which was measured at a wavelength of 610 nm. Log, logstationary (end of log phase), and stationary phase cultures
were sampled. To determine the relationship between gyrB

mRNA expression and growth rate, E. coli W3110 was
cultured in M9 medium (containing 0.5% glucose) at 20,
22, or 25°C. The log phase culture was sampled and 109
cells were collected on a 0.2-μm pore size polycarbonate
membrane filter (Advantec, Japan). The generation time wascalculated using the following formula:
Generation time=log2/μμ=specific growth rate
The specific growth rate was calculated from OD610
measurements (μ=Δln OD610/Δt, where t is time).
2.7. Enumeration of growing bacterial cells
The number of growing bacterial cells was enumeratedusing the method of Kogre et al.14) with minor modifications. E. coli was incubated in LB broth at 37°C. Basedon the growth curve of E. coli, sampling time points wereselected to obtain cultures for total direct count (TDC) and
DVC. The sampling time points were 1.5 h (log phase), 3.5 h(log-stationary phase), and 24 h (stationary phase). Samples
for TDC were fixed with 2% formaldehyde neutral buﬀer
solution (2%; Nacalai Tesque, Japan). Samples for DVC
were incubated in LB broth containing 20 μg mL–1 nalidixic
acid (Wako Pure Chemical Industries Ltd., Japan) at 37°C.
After 1 h, they were fixed with formaldehyde neutral buﬀer
solution. Each fixed sample was stained with 4’,6-diamidino2-phenylindole (DAPI) and counted using an epifluorescencemicroscope (Eclipse 80i; Nikon Co., Japan).
2.8. Sampling of surface river water and river sediment
Surface river water and river sediment was sampled at
Kuwazu from the Inagawa River in Hyogo, Japan. Surface
river water was collected in a sterilized polyethylene
terephthalate copolyester bottle, which was returned to
the lab at 4°C in the dark. River sediment sampling was
performed in triplicate. Sediment samples were transported
to the lab in an ethanol/dry-ice bath. The surface river water
sample was filtered through a 0.2-μm pore size polycarbonate
membrane filter and at least 109 bacterial cells were collectedon the filter. RNA and DNA were recovered from the filteror 2 g (wet weight) of the river sediment. The sediment wasTani et al.

36
Table 2. Expression of gyrB gene in E. coli cellsa
in diﬀerent growth phase

Copy number of gyrB mRNA/cell4.7±2.3
0.015±0.002
<10–6
(n=3)
a
E. coli cells were cultured in LB broth at 37°Cb
During the transition from log phase to stationary phase
Growth phase
Log
Log-stationaryb
Stationary

Table 3. Growing cells in E. coli cellsa in diﬀerent growth phase
Growth phase
Log

Log-stationaryb

Stationary

TDCc 6.6×107±9.3×106 1.2×109±1.2×108 7.0×109±1.2×109
DVCd
98±0.007%
1.0±0.005%
0.64±0.005%(n=3)
E. coli cells were cultured in LB broth at 37°C
During the transition from log phase to stationary phasec
Total direct count (cells/mL)dDirect viable count (percentage of elongated cells per total cells)abFig. 1. Relation between copy number of gyrB mRNA and generation time in E. coli. E. coli cells were cultured in LB broth at
37°C or in M9 medium containing 0.5% glucose at 20°C, 22°C
or 25°C, and were collected at mid log phase. gyrB mRNA were
quantified by real-time RT-PCR. Each blotted dot shows themean of quantified mRNA copy number (n=3), and error bar
shows the standard deviation.

suspended in sterilized deionized water, mixed vigorouslywith a vortex mixer for 30 s, and then sonicated using an
ultrasonic cleaner (Model B3210J; Branson, Danbury, CT)
for 10 min at 120 W. Bacterial cells were trapped on thepolycarbonate filter and enumerated using epifluorescencemicroscopy (E400; Nikon Co., Japan) with UV excitation
after being stained with 1 μg mL–1 of DAPI.

3. Results
The specificity of PCR primers and FRET probes was
experimentally examined in silico. The gyrB gene sequencesof Salmonella spp. were most similar to those of E. coli and
Shigella. The sequences of ESgyrB171F, ESgyrB448R, andESgyrB404 were dissimilar to the corresponding sequencesof Salmonella. ESgyrB376 (3’-labeled FRET probe) matchedperfectly with 8 of the 28 Salmonella DNA gyrase sequences.
ESgyrB404 (5’-labeled FRET probe) had more than two
mismatched bases with all Salmonella DNA gyrase sequences.
PCR using ESgyrB171F and ESgyrB448R was able to amplify
the DNA gyrase gene of E. coli and Shi. sonnei but not thatof Sal. enteritidis, Sal. enterica serovar Choleraesuis, or
V. vulnificus. The copy numbers of gyrB mRNA in an E.
coli cell at the log, log-stationary, and stationary phases in
LB broth at 37°C were 4.7±2.3, 0.015±0.002, and <10–6(below the detection limit, n=3), respectively (Table 2). The
copy number of gyrB mRNA in a log phase cell was 300
times higher than that in a log-stationary phase cell. The

Fig. 2. Expression of gyrB mRNA of E. coli in river surface waterand sediment. Each error bar indicates the standard deviation
derived from triplicate samples.

copy number was below the detection limit in stationary
phase cells. Thus, gyrB mRNA expression levels were high in
growing E. coli W3110 cells, but they declined rapidly after
the cells reached the log phase. When the generation times
were 0.42, 1.5, 5.3, and 9.9 h, the ratios of gyrB mRNA to
DNA were 4.7, 1.5, 0.15, and 0.09, respectively, in E. coli
(Fig. 1).
The ratios of growing bacterial cells to total cells wereenumerated by the DVC method. The ratios of growing E.
coli cells at the log, log-stationary, and stationary phases
were 98±0.007%, 1.0±0.005%, and 0.64±0.005%, respectively (Table 3, n=3). These results agreed with those of gyrB
gene expression.
Following the experiments with standard strain, we evaluated gyrB gene expression in the environment. The numberof bacteria in the surface river water and river sediment was
2.7×107 cells mL–1 and 1.9×109 cells g–1, respectively. The
copy numbers of gyrB for the E. coli-Shigella group were
1.1×104 copies 100 mL–1 for the surface river water sample
and 1.1×104 copies g–1 for the river sediment. The ratio ofmRNA to DNA was 1.3 in the river sediment sample and0.008 in the surface river water sample. The ratio in the sediment was more than 100 times greater than that found in thesurface water (Fig. 2).

Gene expression as an indicator of growth activity

4. Discussion
We found that gyrB mRNA was expressed at high levels
in growing E. coli cells, in which a higher growth rate corresponded to a higher gyrB expression level. DVC and the ratio
of mRNA to DNA were also correlated. These results suggestthat gyrB mRNA expression can be used as an indicator ofbacterial growth activity. The gyrB gene is a housekeeping
gene that encodes for subunit B of bacterial DNA gyrase, andhence, it has an essential role in DNA replication in all bacterial species18). Thus, gyrB can be used to determine growth
activity of bacterial species other than E. coli in naturalenvironments. Only a single copy of the gyrB gene exists in
the genomes of most bacteria. Therefore, the copy numberof the gyrB gene represents the number of bacterial genomes,
which indicates the number of bacteria if the generation time
is more than 1 h. However, multiple copies of genomic DNA
can be found in each cell in more rapidly growing cultures.
The rate of incorporation of a specific compound, such as
thymidine, leucine, or their analogs, is widely used to determine bacterial growth activity7,8). However, this approachrequires samples to be incubated with the appropriate
substance in a closed environment (i.e., a vial), where thebacterial growth activity could change during incubation.
gyrB mRNA expression can be measured without incubation; hence, it can provide a more accurate indicator of insitu bacterial growth activity. The cellular rRNA content
of bacteria reportedly correlates with the bacterial growth
rate5,17). However, the half-life of rRNA in an inactive cell
can be up to several days in natural environments13). The
bacterial rRNA content may indicate the growth rate overthe preceding hours or even over several days, and thus,
gyrB mRNA expression is a more appropriate indicator for
determining in situ bacterial growth activity in a rapidlychanging environment. The frequency of cell division may
accurately indicate the in situ growth activity and growth ratein environments9,15) because incubation is not required for theenumeration of dividing cells. Furthermore, samples used forcounting can be fixed immediately after sampling, which canpreserve the bacterial population in a state that is representative of its state in the original environment. However, counting
bacteria microscopically is often made diﬃcult by the autofluorescence of mineral materials and/or the overlapping ofcells in aggregates, biofilms, and micro-colonies. In contrast,
gyrB mRNA can be measured in various samples if RNA
can be recovered from the sample, including biofilms, soils,
and sediments. The recovery rate of DNA and RNA must be
determined to accurately measure the copy numbers of gyrBDNA and mRNA. In the present study, the recovery rates
were determined by quantifying added DNA (PCR ampliconsof the luciferase gene) and RNA (in vitro transcripts of theluciferase clone), which were used as internal standards.
Neither the luciferase gene nor its transcript was detected in
the river samples (data not shown), and hence, recovery rates
could be accurately determined for each sample. The growth
activity of E. coli-Shigella in surface river water and river37

sediment samples was evaluated by measuring gyrB mRNA
expression. The copy numbers of gyrB for the E. coli-Shigellagroup were 1.1×104 copies 100 mL–1 in the surface riverwater sample and 1.1×104 copies g–1 in the sediment sample.
The ratio of mRNA to DNA was 1.3 in the river sediment
sample and 0.008 in the river surface water sample. The
ratio in the sediment sample was more than 100 times higher
than that in the surface water sample. Thus, river sediment
may be reservoir for the E. coli-Shigella group in naturalenvironments. Microenvironments provided by particulatematter, biofilms, and sediments are reportedly rich in organic
substances and nutrients, and E. coli can grow and survivewell in such environments4,23). However, there has never been
any evidence that E. coli grows in aquatic environments.
The present study showed that E. coli grew in surface riverwater and river sediments and that the growth activity of E.
coli in the river sediment was much higher than that in thesurface river water. These findings suggest that river sedimentis a potential source of E. coli in surface river water, which
is important for understanding the fate of E. coli in aquatic
environments. gyrB is a bacterial housekeeping gene, andmore than 16,000 sequence data entries can now be found
for this gene in DNA databases, which is much more thanother genes related to bacterial growth activity. gyrB is also
useful in the phylogenetic analysis of bacteria18). Phylogenetic inferences based on gyrB sequences are available; thus,
this novel method of measuring gyrB mRNA expression for
determining bacterial growth activity in natural environments
could also be applied to bacterial species other than E. coli
and the Shigella group.

References
1) Artursson, V. and J.K. Jansson. 2003. Use of bromodeoxyuridine immunocapture to identify active bacteria associated witharbuscular mycorrhizal hyphae. Appl. Environ. Microbiol. 69:
6208–6215.
2) Baudisova, D. 1997. Evaluation of Escherichia coli as the mainindicator of faecal pollution. Water Sci. Technol. 35: 333–336.
3) Bernard, L., C. Courties, C. Duperray, H. Schäfer, G. Muyzer,
and P. Lebaron. 2001. A new approach to determine the genetic
diversity of viable and active bacteria in aquatic ecosystems.
Cytometry 43: 314–321.
4) Davies, C.M., J.A.H. Long, M. Donald, and N.J. Ashbolt. 1995.
Survival of fecal microorganisms in marine and freshwater sediments. Appl. Environ. Microbiol. 61: 1888–1896.
5) DeLong, E.F., G.S. Wickham, and N.R. Pace. 1989. Phylogenetic stains: ribosomal RNA-based probes for the cells. Science
243: 1360–1363.
6) Desmarais, T.R., H.M. Solo-Gabriele, and C.J. Palmer. 2002.
Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment. Appl. Envir. Microbiol. 68:
1165–1172.
7) Fuhrman, J.A. and F. Azam. 1980. Bacterioplankton secondary production estimates for coastal waters of British Columbia, Antarctica, and California. Appl. Environ. Microbiol. 39:
1085–1095.
8) Fuhrman, J.A. and F. Azam. 1982. Thymidine incorporation
as a measure of heterotrophic bacterioplankton production inmarine surface waters: evaluation and field results. Mar. Biol.
66: 109–120.

38

Tani et al.

9) Hagström, Å., U. Larsson, P. Hörstedt, and S. Normark. 1979.
Frequency of dividing cells, a new approach to the determination of bacterial growth rates in aquatic environments. Appl.
Environ. Microbiol. 37: 805–812.
10) Hurt, R.A., X. Qui, L. Wu, Y. Roh, A.V. Palumbo, J.M. Tiedje,and J. Zhou. 2001. Simultaneous recovery of RNA and DNA
from soils and sediments. Appl. Environ. Microbiol. 67: 4495–
4503.
11) Iwamoto, T., K. Tani, K. Nakamura, Y. Suzuki, M. Kitagawa,M. Eguchi, and M. Nasu. 2000. Monitoring impact of in situ
biostimulation treatment on groundwater bacterial community
by DGGE. FEMS Microbiol. Ecol. 32: 129–141.
12) Kawai, M., N. Yamaguchi, and M. Nasu. 1999. Rapid enumeration of physiologically active bacteria in purified water used in
the pharmaceutical manufacturing process. J. Appl. Microbiol.
86: 496–504.
13) Kemp, P.F., S. Lee, and J. LaRoche. 1993. Estimating the
growth rate of slowly growing marine bacteria from RNA content. Appl. Environ. Microbiol. 59: 2594–2601.
14) Kogre, K., U. Shimidu, N. Taga, and R.R. Colwell. 1987. Correlation of direct counts with heterotrophic activity for marine
bacteria. Appl. Environ. Microbiol. 53: 2332–2337.
15) Møller, S., C.S. Kristensen, L.K. Poulsen, J.M. Carstensen, and
S. Molin. 1995. Bacterial growth on surfaces: automated image
analysis for quantification of growth rate-related parameters.
Appl. Environ. Microbiol. 61: 741–748.
16) Pernthaler, P., J. Pernthaler, M. Schattenhofer, and R. Amann.
2002. Identification of DNA-synthesizing bacterial cells in
Coastal north sea plankton. Appl. Environ. Microbiol. 68:
5728–5736.
17) Poulsen, L.K., G. Ballard, and D.A. Stahl. 1993. Use of rRNA
fluorescence in situ hybridization for measuring the activity ofsingle cells in young and established biofilms. Appl. Environ.
Microbiol. 59: 1354–1360.
18) Roberts, R.C. and L. Shapiro. 1997. Transcription of genes
encoding DNA replication proteins is coincident with cell cycle

control of DNA replication in Caulobacter crescentus. J. Bacteriol. 179: 2319–2330.
19) Rodriguez, G.G., D. Phipps, K. Ishiguro, and H.F. Ridgway.
1992. Use of a fluorescent redox probe for direct visualizationof actively respiring bacteria. App. Environ. Microbiol. 58:
1801–1808.
20) Rozen, Y. and S. Belkin. 2001. Survival of enteric bacteria inseawater. FEMS Microbiol. Rev. 25: 513–529.
21) Savageau, M.A. 1983. Escherichia coli habitats, cell types,
and molecular mechanisms of gene control. Am. Nat. 122:732–744.
22) Schupp, D.C. and S.L. Erlandsen. 1987. A new method to determine Giardia cyst viability: correlation of fluorescein diacetate
and propidium iodide staining with animal infectivity. Appl.
Environ. Microbiol. 53: 704–707.
23) Sibille, I., T. Sime-Ngando, L. Mathieu, and J.C. Block. 1998.
Protozoan bacterivory and Escherichia coli survival in drinking water distribution systems. Appl. Environ. Microbiol. 64:
197–202.
24) Solo-Gabriele, H.M., M.A. Wolfert, T.R. Desmarais, and C.J.
Palmer. 2000. Sources of Escherichia coli in a coastal subtropical environment. Appl. Envir. Microbiol. 66: 230–237.
25) Tanaka, Y., N. Yamaguchi, and M. Nasu. 2000. Viability ofEscherichia coli O157:H7 in natural river water determined by
the use of flow cytometry. J. Appl. Microbiol. 88: 228–236.
26) Tsai, Y.-L. and B.H. Olson. 1991. Rapid method for direct
extraction of DNA from soil and sediments. Appl. Environ.
Microbiol. 57: 1070–1074.
27) Urbach, E., K.L. Vergin, and S.J. Giovannoni. 1999. Immunochemical detection and isolation of DNA from metabolically
active bacteria. Appl. Environ. Microbiol. 65: 1207–1213.
28) USEPA. 1986. USEPA, Ambient water quality criteria for bacteria-1986. EPA440-584-002. US EPA, Washington, DC.
29) Yamaguchi, N. and M. Nasu. 1997. Flow cytometric analysis
of bacterial respiratory and enzymatic activity in the natural
aquatic environment. J. Appl. Microbiol. 83: 43–52.