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Previous Article | Next Article 
Journal of Bacteriology, March 1999, p. 1733-1738, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Physiological States of Individual Salmonella
typhimurium Cells Monitored by In Situ Reverse
Transcription-PCR
Kim
Holmstrøm,1
Tim
Tolker-Nielsen,2 and
Søren
Molin2,*
Biotechnological Institute, DK-2970
Hørsholm,1 and Department of
Microbiology, The Technical University of Denmark, DK-2800
Lyngby,2 Denmark
Received 8 July 1998/Accepted 25 November 1998
 |
ABSTRACT |
The possibility of using levels of specific mRNAs in individual
bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of groEL and tsf mRNAs
per cell in Salmonella typhimurium cells in different
physiological states were obtained by Northern analysis. The average
number of groEL mRNAs per cell was estimated to be 22 in
fast-growing cultures and 197 in heat-shocked cultures. The average
number of tsf mRNAs per cell was estimated to be 37 in
fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing
cultures. The potential of mRNA-targeted in situ reverse
transcription (RT)-PCR to monitor quantitatively different levels of
groEL and tsf mRNA in individual cells and thus monitor both specific gene induction and general growth
activity was assessed. Neither groEL nor tsf
mRNA was present in stationary-phase cells, but it was shown
that stationary-phase cells contain other RNA species at high levels,
which may provide a possibility for monitoring directly
stationary-phase individual cells by the use of in situ RT-PCR.
The outcome of the in situ RT-PCR analyses indicated that
a population of fast-growing cells is heterogeneous with respect to
groEL mRNA single-cell contents, suggesting a cell-cycle-controlled expression of groEL in S. typhimurium, whereas a fast-growing culture is homogeneous
with respect to tsf mRNA single-cell contents,
suggesting that the level of tsf mRNA is relatively
constant during the cell cycle.
| |
INTRODUCTION |
Detection of bacteria and monitoring
of their activities in situ are of great importance for understanding
their role and performance in the environment and are also useful in
areas of microbiology such as public health, food technology, and the
pharmaceutical industry. In addition, the monitoring of bacteria with
information of their physiological states may allow prediction of, or
even design of, biological system properties such as productivity, biomass turnover, substrate utilization, or pollutant degradation.
Over the last decade, major advances have been achieved in the
application of nucleic-acid-based methods for species-specific detection of microorganisms. The phylogenetic signature
sequences of rRNA were recognized early as targets for
hybridization (27, 28). The natural amplification of
rRNA normally results in excellent sensitivities of hybridization
assays, facilitating, for example, in situ identification of individual
cells by use of fluorochrome-labeled oligonucleotides (1,
6). Although the correlation between bacterial ribosome content
and growth rate (31) is reflected when quantitative rRNA
hybridization is applied to bacteria with different growth rates
(6, 26), the content of rRNA in individual cells
cannot in general be used to directly estimate the actual growth
activity of the bacteria (33).
A number of methods have been applied to monitor the activities of
bacteria at the single-cell level. Examples include monitoring of
substrate uptake (14), growth ability (18),
intracellular enzyme activity (7), respiration
activity (15, 30, 38), and membrane potential activity
(17, 23). All of these methods give information about
the general physiological state of the bacteria, but they cannot give
detailed information regarding which genes the bacteria expressed at
the time of sampling. Recently, methods for the detection
of specific mRNAs in individual bacterial cells have been developed
(11-13, 34). Since mRNAs are intermediates in gene
expression, the detection of specific mRNA in individual cells may
provide many-sided information on the physiology of bacteria in situ.
In the present report we show that both specific gene expression and
general growth activity can be monitored at the single-cell level by
the use of mRNA-targeted in situ reverse transcription (RT)-PCR.
| |
MATERIALS AND METHODS |
Strains, growth, and heat shock conditions.
Salmonella
typhimurium LT2 (21) was used as model organism in
these studies. S. typhimurium SL4213 F' lacY
(34) was used in the estimation of the average number of
specific mRNAs per cell. Luria-Bertani (LB) broth (4)
was used as growth medium throughout, except for in the RNA arbitrarily
primed (RAP) PCR experiment, where AB minimal medium (5)
supplemented with 0.2 or 0.02% glucose was used.
The SL4213 F' lacY strain was grown at 37°C for at least
10 generations in the presence of 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) before the
exponential-phase culture was fixed for lac mRNA-targeting Northern analysis and the determination of CFU.
Mid-log-phase S. typhimurium LT2 cells cultivated at
37°C were subjected to heat shock by fourfold dilutions in LB broth
preheated to either 45 or 52°C. Stationary-phase cells were
established by continued incubation for up to 20 h after the
exponential growth had leveled off.
Determinations of CFU were done by diluting the samples in 0.9% NaCl
followed by plating on LB agar and counting of colonies.
Primers and radiolabeled probes.
Primers were used for PCR
generation of probes for Northern analyses and for seminested in situ
RT-PCR detection of mRNA. In addition, a single arbitrarily
chosen primer, cST6 (5'-CTTTGTCGTTTTCACCTCGCTG-3'), was used
in RNA arbitrarily primed PCR. Based on the sequence of the
S. typhi groEL gene (22) and the
Escherichia coli tsf gene (2), two sets of
primers for seminested PCR amplification of the putative S. typhimurium groEL and tsf genes were designed: groELf
(forward-primer), 5'-TCCGCTAACTCCGACGAAAC-3'; groELr
(reverse primer), 5'-AGCAACCACGCCTTCTTCTAC-3'; groELi
(internal primer), 5'-TCGCTTCTTCAATCTGCTGAC-3'; EF-Tsf
(forward primer), 5'-CATATGGCCCCCTTTTTCACTTTT-3'; EF-Tsr
(reverse primer), 5'-TGCGGCCTTCAACCATTTTCT-3'; and
EF-Tsi (internal primer), 5'-CTCTTCGTCAGCGCCTTTAGCA-3'.
The two primers groELi and EF-Tsi were labelled with biotin
at the 5' end during the automated synthesis and were subsequently
purified by reversed-phase high-performance liquid chromatography. All
primers were tested by PCR with purified S. typhimurium
LT2 DNA as the template and were found to give PCR products of the
expected sizes: 789 bp with groELf plus groELr, 619 bp with groELf plus
groELi, 732 bp with EF-Tsf plus EF-Tsr, and 566 bp with EF-Tsf plus
EF-Tsi. The PCR products of groELf plus groELr and EF-Tsf plus EF-Tsr
were used as probes in Northern analyses to target the groEL
and tsf mRNA molecules, respectively. An 1,167-bp
fragment that was PCR generated with previously described primers
(lacZf and lacZr) (34) was used to target lac
mRNA from the S. typhimurium F' lacY
strain in Northern blots.
Fifty nanograms of all probes for Northern blot analysis were random
primer labeled with Ready-To-Go DNA Labeling Beads and [
-32P]dCTP (3,000 Ci/mmol; Amersham Pharmacia Biotech,
Hørsholm, Denmark) according to the manufacturers' instructions. The
labelled probes were separated from unincorporated
32P-labelled nucleotides by gel filtration in a NICK column
(Amersham Pharmacia Biotech), and the efficiency of labeling was
subsequently monitored by thin-layer chromatography.
RNA isolation and Northern analyses.
Total RNA was extracted
from 5 × 108 to 5 × 109 cells as
previously described (9). Cells were harvested and
resuspended in 890 µl of extraction mixture I (80 mM Tris-HCl, pH
7.5; 10 mM MgCl2; 10 mM 2-mercaptoethanol) followed by the
addition of 1,110 µl of extraction mixture II (0.9% sodium dodecyl
sulfate [SDS], 0.41 mg of proteinase K per ml, 18 mM EDTA, 3.6 mM
1.10-phenanthroline, 0.36 mg of heparin per ml) and incubation at
37°C for 20 min and 65°C for 10 min. One volume of
phenol-chloroform-isoamyl alcohol (25:24:1) was added to the extraction
mixture, and the resulting aqueous phase was reextracted once with
phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform.
RNA was precipitated with 0.3 M sodium acetate and a 2.5 volume of 96%
ethanol overnight at 
20°C, washed once in 70% ethanol, and allowed
to dry before resuspension in 50 µl of diethylpyrocarbonate
(DEPC)-treated distilled H2O. Isolated RNA was kept at
80°C until used.
Separation of RNA fragments in 1.2% formaldehyde agarose gels and
subsequent blotting onto Hybond-N+ nylon membranes
(Amersham Pharmacia Biotech) was done as previously described
(8). All prehybridizations and hybridizations were performed
in 0.5 M sodium phosphate (pH 7.2) with 7% SDS at 65°C. Prehybridizations were performed for 3 h, followed by substitution of the prehybridization mixture with fresh prewarmed (65°C)
hybridization mixture with heat-denatured radiolabeled probe added.
Hybridizations were performed overnight. Washes (three times for 15 min) were done in 20 mM sodium phosphate (pH 7.2) with 1% SDS at
65°C. The washed membrane filters were subsequently autoradiographed
and subjected to quantification of radioactive signals from specific bands by using an Electronic Autoradiography Instant Imager device (Packard Instrument Company, Meriden, Conn.).
Estimation of average numbers of mRNAs per cell.
In
order to quantify the number of groEL and tsf
mRNAs per cell, the same amount of a reference probe (the
lacZ mRNA targeting probe) and the two probes for
detection of groEL and tsf and mRNA were
radiolabeled with 32P to the same specific activity (as
determined by thin-layer chromatography). The probes were hybridized to
membranes that contained total RNA from the same number of cells in
each lane. With the assumption that all of the probes hybridize with
equal efficiency to complementary targets, the accumulated counts over
time, obtained by quantification of the radioactivity emitted from the
specific bands on the hybridized membrane by use of the Instant Imager
device, could be quantitatively compared. Counts from the
lacZ probe were always set to 20 mRNA per cell (see
reference 34 for a detailed explanation on the calculation of this number) and thus it was possible to estimate the
average numbers of the different mRNAs per cell by taking into
account the differences in the size of the probes. Estimates of
mRNA numbers per cell represent the average of two independent quantitative Northern analyses.
mRNA-targeted in situ RT-PCR.
In situ RT-PCR was
done essentially as described earlier (34). Since prolonged
heat treatments of the cells prior to fixation made it possible to
amplify intracellular DNA (data not shown), a DNase I treatment was
performed after cell membrane permeabilization in order to avoid
signals attributable to DNA amplification. Since biotin-labeled
internal primers were used (instead of fluorescein-labeled internal
primers), the biotinylated PCR products inside the cells were detected
by using a streptavidin-horseradish peroxidase conjugate (sa-HRP)
(Dupont/NEN Research Products) in combination with the fluorescence-based tyramide signal amplification system (Dupont/NEN Research Products).
Microscopy and image processing.
Epifluorescence and
phase-contrast microscopy and processing of digitized charge-coupled
device-camera-captured images was done as described previously
(34).
RAP-PCR.
RAP-PCR was performed essentially as described by
McClelland and Welsh (24). Approximately 200 and 30 ng RNA
from 106 exponentially growing and established
stationary-phase cells, respectively, were initially subjected to
treatment with 10 U of RNase-free DNase I (Promega) for 4 h at
37°C in 100 µl of DNase I-buffer (40 mM Tris-HCl, pH 7.9; 10 mM
NaCl; 6 mM MgCl2; 10 mM CaCl2) to remove traces
of DNA. Phenol-chloroform-isoamyl alcohol and chloroform extractions
were performed, and the RNA was precipitated with ethanol as described
earlier. The RNA pellet was resuspended in 10 µl of DEPC-treated
distilled water, and 10 µl of 2× "first-strand reaction mix"
(100 mM Tris-HCl, pH 8.3; 100 mM KCl; 8 mM MgCl2; 20 mM
dithiothreitol; 0.4 mM concentrations of dATP, dGTP, dCTP, and dTTP; 20 µM cST6; 200 U of Moloney murine leukemia virus [Mo-MuLV] RT [Life
Technologies]) was added. Parallel negative controls consisted of
exactly the same components except for the Mo-MuLV RT enzyme. Reactions
were incubated in a Perkin-Elmer 9600 thermal cycler under the
following conditions: ramp over 5 min to 37°C and holding this
temperature for 10 min, followed by 2 min at 94°C and cooling to
4°C for 5 min. To each reaction, 20 µl of 2× "second-strand
reaction mix" (10 mM Tris-HCl, pH 8.3; 25 mM KCl; 2 mM
MgCl2; 2 µCi of [-32P]dATP [3,000
Ci/mmol; Amersham Pharmacia Biotech]; 4 U of Taq DNA
polymerase [Perkin-Elmer]) was now added, and the reactions were
cycled through 1 low-stringency step (1 min at 94°C, 5 min at 40°C,
and 5 min at 72°C) followed by 35 high-stringency steps (1 min at
94°C, 1 min at 57°C, and 1 min 72°C) in the thermal cycler. Next,
4 µl of each reaction was mixed with 4 µl of loading buffer (95%
formamide; 20 mM EDTA; 0.05% bromophenol blue; 0.05 xylene cyanol),
heated to 94°C for 2 min, and then cooled to 4°C for 5 min.
Finally, 3 µl of each reaction was loaded onto a 6% polyacrylamide-50% urea sequencing gel, and electrophoresis was performed in 1× TBE buffer (Tris borate-EDTA) at 55 W for 2.5 h.
The gel was dried and exposed on a Kodak Biomax MR film overnight.
| |
RESULTS AND DISCUSSION |
Monitoring of cells expressing a heat-shock-induced gene.
We
have previously demonstrated the ability of mRNA-targeted
in situ RT-PCR to monitor the presence or absence of lac
mRNA in S. typhimurium (34, 35).
However, in order to obtain detailed information about the growth
physiology of bacteria at the single-cell level, it will be necessary
to distinguish between different levels of specific mRNAs instead
of just their presence or absence. With the aim of performing
quantitative determinations of specific mRNA levels by use of in
situ RT-PCR, we chose to target the groEL mRNA in
S. typhimurium cells in different physiological states. The groEL gene is part of the 
32-directed
heat-shock regulon, it encodes a molecular chaperone, it is expressed
at a low level in growing bacteria, and it is induced when the bacteria
are exposed to higher temperatures (10). As model
cellular physiological states we chose exponential growth at 37°C,
heat shock at 45°C, heat-induced death at 52°C, and stationarity at
37°C.
Although the regulation of the groEL gene, as described
above, is known, the number of groEL mRNAs per cell in a
given physiological state has, to the best of our knowledge, not been
reported. In the present study it was important to have
information about the number of groEL mRNAs in
S. typhimurium cells in different physiological states. Therefore, Northern analysis was performed on samples containing equal numbers of cells that were either growing at 37°C,
heat shocked at 45°C or 52°C, or in stationary phase. In order to
estimate the number of groEL mRNAs per cell in the
different conditions, samples of fully IPTG-induced S. typhimurium F' lacY cells, in which the number of
lac mRNAs per cell had been determined to be
approximately 20 (34), was included in the Northern
analysis. Figure 1 shows the result of
the groEL targeted Northern blot. By quantification of the
hybridization signals from the groEL mRNA band in the
different lanes and the lac mRNA band in the reference
lane, the number of groEL mRNAs per cell was estimated (Table 1). When the cells grew
exponentially at 37°C they had an estimated average of approximately
22 groEL mRNAs. At 15 min after the shift to 45°C, the
number of groEL mRNAs per cell had increased
approximately nine times. At 6 min after the shift to 52°C, this
increase was approximately 2.5 times. The level of groEL
mRNA at 45°C decreased after the initial burst to a level after
1 h, which was about two times higher than the initial level, in
agreement with observations of the transcription of
32-controlled heat shock genes in E. coli
(19, 37). The cells at 45°C, however, were not able to
maintain the high number of groEL mRNAs per cell;
although they were alive and growing (see Fig. 2), they contained only
seven groEL mRNAs per cell 3 h after the
temperature shift. When the cells were subjected to 52°C they died
(Fig. 2), and the initially increased
number of groEL mRNAs per cell started to decrease
earlier than in cells at 45°C. When the bacteria entered stationary
phase a decrease in the level of groEL mRNAs per cell
was observed. In the early stationary phase (approximately 2 h
after exponential growth ceased) the groEL mRNA was
present in an average number of three per cell, and in the established
stationary phase (approximately 20 h after exponential growth
ceased) the cells were depleted of groEL mRNA.
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FIG. 1.
Northern analysis of groEL mRNA in
S. typhimurium LT2 (performed as described in Materials
and Methods). The Northern blot contains RNA samples from (A)
exponential-phase cells (lane 1), cells incubated at 52°C for 6 min
(lane 2), 15 min (lane 3), 60 min (lane 4), 120 min (lane 5), and 180 min (lane 6) and cells incubated at 45°C for 6 min (lane 7), 15 min
(lane 8), 60 min (lane 9), and 180 min (lane 10) and (B)
exponential-phase cells (lane 1), early-stationary-phase cells (lane
2), and established-stationary-phase cells (lane 3). The size of the
major transcript is given at the arrow. The sizes of the major
transcripts are given at the arrows.
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FIG. 2.
Determination of cfu of heat-shocked S. typhimurium LT2 cells expressed as the percentage of survival
relative to the CFU number just before heat shock. Heat shock and CFU
determinations were performed as described in Materials and Methods.
The solid line corresponds to heat shock at 45°C, and the dotted line
corresponds to heat shock at 52°C. The standard deviations of the
measurements are indicated by vertical lines.
|
|
In parallel with the samples for Northern analysis, samples were also
taken for groEL mRNA-targeted in situ RT-PCR. The in situ RT-PCR was performed with reporter-molecule-labeled primers, and intracellular reporter molecules were subsequently detected by use
of a fluorogenic assay. As shown in Fig.
3, it was indeed possible to visualize
differences in groEL mRNA levels between uninduced cells
(Fig. 3A), heat-shock-induced cells (Fig. 3B), and RNase-treated
heat-shock-induced cells (Fig. 3C). The population of cells that grew
exponentially at 37°C prior to in situ RT-PCR showed
heterogeneity with respect to groEL mRNA-conferred
fluorescence after in situ RT-PCR (Fig. 3A). This signal
heterogeneity was not caused by differences in the
permeabilization of the individual cells, since cells from the
same culture subjected to tsf mRNA-targeting in
situ RT-PCR were homogeneous with respect to fluorescence intensity (see below). We therefore assume that the observed heterogeneity was
caused by different levels of groEL mRNA in the
individual cells, which may be the result of cell-cycle-controlled
expression of groEL in growing cells. A cell cycle temporal
regulation of the groEL operon has previously been observed
for the gram-negative bacterium Caulobacter crescentus
(3). The estimate of 22 groEL mRNAs per cell
in growing cells should therefore be considered as an average covering
a spectrum of different numbers in individual cells, depending on their
cell age at the time of sampling. The fact that we observed cells from
the uninduced culture that were comparable in signal intensity to cells
that had been RNase treated could mean that cells at a certain point in
the cell cycle do not contain groEL mRNA or,
alternatively, that the groEL mRNA content in these
cells was below the detection limit of the
groEL-targeted in situ RT-PCR procedure.
Heterogeneity with respect to cell fluorescence was also observed when
cells from the culture growing slowly at 45°C for 1 h after the
shift from 37°C was subjected to groEL-targeting in situ
RT-PCR (data not shown). In the induced sample shown in Fig. 3B,
the signal from a few cells was not distinguishable from background
signals, and these cells may be dead. Evidence has previously been
presented, that about 5% of the cells in a growing Salmonella culture were dead cells (20).
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FIG. 3.
Detection of groEL mRNA in S. typhimurium LT2 cells. Cells were treated as described in
Materials and Methods. Phase-contrast photomicrographs (top panels) and
epifluorescence photomicrographs (bottom panels) of the same fields are
shown. (A) Mid-log phase cells. (B) Cells heat shocked at 45°C for 15 min. (C) Cells heat shocked at 45°C for 15 min and RNase-treated
prior to the in situ RT-PCR.
|
|
Previously, we showed that in situ RT-PCR could be used to
distinguish between lac-mRNA-containing and
lac-mRNA-free cells in a nonquantitative manner, but
it was also suggested that a less-sensitive in situ
RT-PCR procedure might be obtained by adjusting one or more of
a number of PCR parameters (34). The groEL primer set and template used in the present study may result in a
less-efficient PCR performance compared to that used previously for
lac mRNA detection. It is possible that the
groEL-targeted in situ RT-PCR used here did not reach
the point of saturation and that therefore differences in
groEL mRNA contents could be visualized.
Monitoring of growing cells.
To determine if single bacterial
cells are growing or not is of great importance when bacterial
communities are characterized. The ability of mRNA-directed
in situ RT-PCR to monitor the growth of bacteria was tested
by targeting the tsf mRNA encoding elongation factor EF-Ts. The tsf gene is under stringent control and is
coordinated with the synthesis of ribosomal proteins as a function of
growth rate in E. coli (25). As model
physiological states, we chose fast exponential growth at 37°C,
slow growth at 45°C, stationarity at 37°C, and heat-induced death
at 52°C.
Numbers of tsf mRNAs per cell in the different
physiological states were estimated by Northern analysis, again by
using the content of lac mRNA in fully IPTG-induced
S. typhimurium F' lacY cells as a reference
sample. Figure 4 shows the
tsf-targeted Northern blots, and the estimated numbers of
tsf mRNA molecules per cell are presented in Table
2. When the cells were growing
exponentially at 37°C (doubling time of 23 min) they contained on
average ca. 37 tsf mRNAs. After the shift to 45°C the
cells grew with a much increased doubling time (ca. 90 min) (see Fig.
2) with an approximate eightfold reduction of the tsf
mRNA level. When shifted to 52°C the level of tsf
mRNA per cell declined rapidly, and after 3 h the cells were
depleted of tsf mRNA. When the bacteria entered the
stationary phase, a fast decrease in the level of tsf
mRNAs per cell was observed. Already in the early stationary phase
(approximately 2 h after leaving the exponential-growth phase) the
culture was largely depleted of tsf mRNA.
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FIG. 4.
Northern analysis of tsf mRNA in
S. typhimurium LT2 cells (performed as described in
Materials and Methods). The Northern blot contains RNA samples from (A)
exponential-phase cells (lane 1); cells incubated at 52°C for 6 min
(lane 2), 15 min (lane 3), 60 min (lane 4), 120 min (lane 5), and 180 min (lane 6); and cells incubated at 45°C for 6 min (lane 7), 15 min
(lane 8), 60 min (lane 9), and 180 min (lane 10) and (B)
exponential-phase cells (lane 1), early-stationary-phase cells (lane
2), and established-stationary-phase cells (lane 3). The size of the
major transcript is given at the arrow.
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|
The cells in the different physiological states were subjected to
tsf mRNA-targeted in situ RT-PCR. Cells that were
growing at 37°C prior to sampling were brightly fluorescent after in
situ RT-PCR (Fig. 5A), indicating the
presence of high levels of tsf mRNA. Cells that had been
growing slowly at 45°C for 1 h after the shift from 37°C
fluoresced less intensely after being subjected to tsf
mRNA-targeted in situ RT-PCR (Fig. 5B), suggesting again that the in situ RT-PCR can be used at least
semiquantitatively to distinguish in this case approximately
eightfold differences in the mRNA content. Cells in the
early stationary phase showed only background fluorescence after being
subjected to the tsf mRNA-targeting in situ RT-PCR
(Fig. 5C), indicating, in accordance with the Northern blot analyses,
that these cells were almost completely depleted of tsf
mRNAs already at this point.
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FIG. 5.
Detection of tsf mRNA in S. typhimurium LT2 cells. Cells were treated as described in
Materials and Methods. Phase-contrast photomicrographs (top panels) and
epifluorescence photomicrographs (bottom panels) of the same fields are
shown. (A) Mid-log phase cells. (B) Cells heat shocked at 45°C for 60 min (slow growing). (C) Cells in the early stationary phase (2 h after
exponential growth ceased).
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In contrast to what we observed with the groEL
mRNA content in growing cells, the population of fast-growing
cells appeared to be homogeneous with respect to tsf
mRNA content (Fig. 5A), suggesting that the number of
tsf mRNAs per cell is relatively constant during the
cell cycle. The apparent heterogeneity with respect to single-cell
tsf mRNA contents in the population of cells that were
growing slowly at 45°C (Fig. 5B) may be a consequence of the culture
not being balanced only 1 h after the shift from 37°C.
Monitoring of stationary-phase cells.
It is of general
interest to identify cells that are alive but in stationary phase in
complex microbial communities. In order to do this by in situ
RT-PCR, a gene which is specifically expressed in the stationary
phase should be targeted. Since both the groEL and
tsf mRNAs were absent in stationary-phase cells, it was
of interest to determine if any mRNAs at all are present in
stationary-phase cells. For this purpose, we applied the
so-called RAP-PCR procedure (36) to samples containing
equal numbers of either mid-log-phase or stationary-phase S. typhimurium cells. Each lane in Fig.
6 contains a RAP-PCR profile
corresponding to the presence of a subgroup of RNA molecules PCR
amplified with an arbitrarily chosen primer. Corresponding bands in
different profiles can be compared quantitatively, assuming that the
amount of total RNA in each reaction does not interfere with the
amplification efficiency of individual RNA targets. The RAP-PCR profile
of stationary-phase cells contain some of the bands also seen in the
RAP-PCR profile of exponential-phase cells, and some of the bands have
the highest intensities in the RAP-PCR profile of stationary-phase
cells (Fig. 6). This indicates that some RNA targets are present in
equal amounts per cell in growing and stationary-phase cells and that some RNA targets are present in higher numbers in the stationary-phase cells than in the growing cells. Besides opening the possibility of monitoring stationary-phase cells by the use of in situ RT-PCR, this holds promise for using specific mRNAs as viability markers for single cells, since mRNA is assumed to be depleted in dead cells (16, 29, 32). Work is currently in progress to
identify some of the mRNAs which are present in stationary-phase
cells.
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FIG. 6.
RAP-PCR of total RNA from approximately 106
S. typhimurium LT2 cells in exponential growth (lane 1)
and in established stationary phase (lane 2). See Materials and Methods
for more details on the procedure. Examples of bands potentially
originating from RNA species, which are present in higher number in the
stationary phase than in the mid-log phase (a), which disappear in
stationary phase (b), and which seem to be present in the same amounts
in both stationary and exponential phase (c), are indicated by
arrows.
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Concluding remarks.
We have shown that levels of specific
mRNAs in single cells, monitored by in situ RT-PCR, can be used
as indicators of the physiological state of individual bacteria. The
groEL and tsf mRNAs belong to different
classes of single-cell physiology indicators. The groEL
mRNA gives information about a specific physical condition of the
bacterium. Analogously, genes induced by other physical stress
conditions and genes induced by starvation for a specific compound also
belong to this class of indicators. The tsf mRNA provides information about the growth activity of the bacterium, but it gives no detailed information about the local environment sensed
by the bacterium. This class of single-cell physiology indicators also
includes mRNAs that are synthesized preferentially in
stationary-phase cells. We believe that determinations of specific mRNA contents as indicators of single-cell physiology and
performance will become a very powerful tool in studies of the
biology of microorganisms in complex natural communities.
| |
ACKNOWLEDGMENTS |
The expert technical assistance of Torben Kallesøe in performing
the RAP-PCR analysis is acknowledged.
This work was supported in part by a grant from the Danish Research and
Development Program for Food Technology (FØTEK2).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Microbiology, Bldg. 301, The Technical University of Denmark,
DK-2800 Lyngby, Denmark. Phone: 45-45252513. Fax: 45-45887328. E-mail: sm{at}im.dtu.dk.
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Journal of Bacteriology, March 1999, p. 1733-1738, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
