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Department of Biological Sciences and Program in Biochemistry, Smith College, Northampton, MA 01063, USA
Correspondence
Christine A. White-Ziegler
cwhitezi{at}smith.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GEO accession number for the microarray data reported in this paper is GSE9197.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Mahan et al., 1996; Mekalanos, 1992).
Bacteria have the ability to respond to temperature as a primary cue to regulate gene expression. For mesophilic organisms, the most well studied are the reactions to temperatures at the limit of growth for these bacteria – the heat- (42 °C) and cold-shock (15 °C) responses (reviewed by Gross, 1996; Phadtare et al., 2000; Yura et al., 2000). Recently, microarray studies from our laboratory and others have focused on genes that are preferentially expressed at 37 °C, identifying genes regulated by this host cue that may ensure more efficient colonization (Brooks et al., 2003; Han et al., 2004; Motin et al., 2004; Revel et al., 2002; Smoot et al., 2001; White-Ziegler et al., 2007). In these studies, the comparison is made to growth at lower temperatures. Adaptation to growth at the lower temperature is particularly relevant in Yersinia pestis and Borrelia burgdorferi as it mimics the environment of an insect host (Brooks et al., 2003; Han et al., 2004; Motin et al., 2004; Revel et al., 2002). During its life cycle, E. coli is likely to encounter shifts to lower temperatures, either long-term or transient, that are similar to ambient indoor room settings (18–23 °C). An understanding of how E. coli adapts to this temperature is particularly important in medical and food industry settings where prevention of bacterial contamination is imperative.
One well understood model of gene regulation by low temperature (25 °C) is through the action of the general stress response sigma factor RpoS and the small regulatory RNA DsrA. Sledjeski et al. (1996) demonstrated that low temperature causes the increased expression of RpoS under low-temperature conditions during exponential phase in both rich (LB) and minimal (M63) medium through the action of the small regulatory RNA DsrA. Transcription of dsrA is increased at low temperature and DsrA interacts with the rpoS mRNA to alter its secondary structure to allow more efficient translation of the rpoS mRNA (reviewed by Lease & Belfort, 2000; Repoila et al., 2003). Thus, transcription of RpoS-dependent genes is expected to be induced at low temperature in exponential phase, although this model has only been directly proven for a few genes, including csgB, csgA, csgD (Brown et al., 2001; Olsen et al., 1993b) and dsrB (Sledjeski et al., 1996).
In this study, microarray studies using E. coli K-12 MC4100 were completed and demonstrate that 297 genes, approximately 7 % of the genome, are more highly expressed at 23 °C compared to growth at 37 °C. Approximately 40 % of the genes preferentially expressed at 23 °C are RpoS-controlled genes, broadly supporting and expanding the model that low temperature is a primary environmental cue that triggers the general stress response. Of the genes with increased transcription at 23 °C, two categories of genes were specifically noted – those associated with cold shock and biofilm development. The former set indicate that similar mechanisms used to adapt to a sudden decrease in temperature (>15 °C) are also used for long-term adaptation to growth at ambient conditions, whereas the latter set would suggest that temperature is an environmental cue that might impact biofilm development. In this study, the effect of low temperature was investigated for its effect on gene expression as well as its physiological effects on growth, cold-shock viability and biofilm formation. Given the number of genes controlled by RpoS that are expressed at 23 °C, the impact of temperature was tested in the wild-type and in rpoS and dsrA mutant strains to assess the contribution of these regulatory factors to the overall thermoregulatory response.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Luria–Bertani (LB) and M9 minimal media and antibiotics were prepared as described by Miller (1972) and Silhavy et al. (1984). rpoS : : Tn10 (from RH90) and the dsrA : : cat (from SG12067) mutations were introduced into DL1504 by P1 transduction (Silhavy et al., 1984) to create DL3106 and CWZ458, respectively (Table 1).
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, 2007). An inoculum from a 37 °C-grown single colony was used to initiate parallel 37 °C and 23 °C cultures. The cells from these cultures were harvested at equivalent optical densities after approximately 9–11 generations of growth in early mid-exponential phase (OD600=0.2–0.6). Cell pellets were subsequently frozen and stored at –80 °C for RNA isolation. Identical methods were used for cells grown in LB or M9 glucose media. For temperature shift experiments in M9 glycerol, an initial culture was inoculated as described above and grown at 37 °C to mid-exponential phase. The culture was diluted 1 : 1 in fresh M9 glycerol and grown for an additional 45 min at 37 °C. The culture was then shifted to 23 °C and samples were collected at 0, 1, 2, 5, 7 and 11 h. For time points of 2 h and longer, the starter culture was additionally diluted in M9 glycerol to ensure the collection of cells within the exponential phase.
For growth curve analyses, an initial culture was inoculated as described above and grown at 37 °C to early exponential phase. The culture was subsequently shifted to 23 °C and spectrophotometer readings were taken at time points after the shift. The generation time for each strain was determined as described by Miller (1972).
For the cold-shock assay, an initial culture was inoculated as described above and grown at 37 °C to early exponential phase. A tenfold dilution series of each culture was performed in microtitre plates in M9 glycerol salts and 5 µl of each dilution was plated on M9 glycerol plates for determination of the number of c.f.u. ml–1. A set of plates was incubated at 23 °C for immediate colony outgrowth (t=0). The remaining plates were incubated at 4 °C in a humidified chamber from 1 to 6 days and subsequently incubated at 23 °C for colony outgrowth.
RNA isolation.
For microarray analyses, RNA was isolated by phenol/chloroform extraction as described previously (White-Ziegler et al., 2007). For quantitative real-time RT-PCR (qRT-PCR) experiments, RNA isolation was done using Qiagen RNeasy Mini columns as described previously (White-Ziegler et al., 2007). RNA concentrations and purity were determined by spectrophotometer readings. Isolated RNAs were stored at –80 °C until used.
cDNA synthesis, labelling and hybridization.
Synthesis, labelling of cDNA with Cy3/Cy5 and hybridization was performed using the 3DNA Array 350 RP Expression Array Detection kit as described previously (White-Ziegler et al., 2007). cDNA for each condition was prepared from 2 µg total RNA. cDNA from cultures grown at both temperatures (37 and 23 °C) was co-hybridized to slides containing full-length PCR products from all 4290 annotated ORFs in E. coli MG1655. Slides were produced by the University of Wisconsin-Gene Expression Center (www.biotech.wisc.edu/GEC/) and obtained at a reduced cost through the Genome Consortium for Active Teaching (www.bio.davidson.edu/projects/gcat/gcat.html).
Microarray data analysis.
Five slides were used in the analysis with cDNAs, representing three independent growth experiments and two technical replicates. Hybridized slides were scanned and the data were analysed as described previously (White-Ziegler et al., 2007). Significance analysis was completed as a one-class response using Significance Analysis of Microarrays (SAM) (Tusher et al., 2001) with 
=0.65 and a median false discovery rate of 1 %. An ORF was considered temperature-regulated if it demonstrated a statistically significant change in expression greater than 1.7-fold.
qRT-PCR.
Reactions were completed using the SYBR Green One Step qRT-PCR kit (Invitrogen) as described previously (White-Ziegler et al., 2007). All reactions were performed in triplicate, with no reverse transcriptase and no RNA controls run for each RNA sample to detect DNA contamination and reagent contamination, respectively. All reactions were normalized by using the same amount of total RNA (50 ng) in each reaction. Relative levels of gene expression and error analysis were calculated as described previously (Livak & Schmittgen, 2001; White-Ziegler et al., 2007)
Biofilm formation assays.
The biofilm assay was modified from a method described by O'Toole & Kolter (1998) with the following changes. Wells of 96-well polystyrene microtitre plates were inoculated with 200 µl bacterial culture that was diluted to a calculated starting OD600 of 0.003 (approx. 1 : 1000 dilution of an overnight culture) in fresh M9 glycerol medium. The plates were incubated in a humidified chamber for varying times at 37 or 23 °C. At a given time point, 100 µl of the culture was removed to a separate plate and the OD600 was recorded as a measure of planktonic growth. To the remaining 100 µl in the original microtitre plate, 100 µl M9 salts and 25 µl 1 % crystal violet was added and allowed to stain for 15 min. The plates were subsequently rinsed vigorously with water. To quantify biofilm formation, 250 µl 95 % ethanol was pipetted into each well and 125 µl was removed to a separate plate where the OD600 was recorded as a measure of biofilm formation. Differences in biofilm formation were determined to be statistically significant (P<0.05) using two-way analysis of variance (ANOVA) using STATA SEM software (StataCorp). Data represent the mean -fold change determined from at least three independent experiments.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) whereas 126 genes were found to be more highly expressed at 37 °C (White-Ziegler et al., 2007).
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). The genes span the different COG functional categories with none of the known functional classes particularly dominating the response. For each functional COG category, the number of genes in each category ranges between 0 and 8 % of the total genes (297) identified. The largest subset of genes (36 %, 107/297) are not categorized in a particular COG functional group. Strikingly, approximately half of the genes more highly expressed at low temperature are hypothetical or of unknown function. Thus, there are a significant number of uncharacterized genes whose expression is increased in response to low temperature and are likely to be important in adaptation to this environmental change.
Low temperature increases expression of RpoS-controlled genes
Comparison of our gene list with other published sources showed that 122 genes with increased expression at 23 °C are known to be RpoS-controlled in response to at least one other environmental stress (Table 2
), with the majority (92 genes) overlapping a core set of genes known to be induced by three different environmental stresses (high osmolarity, low pH and stationary-phase growth) (Weber et al., 2005). Included within our dataset are four genes previously shown to specifically be induced by low temperature in an RpoS-dependent manner – csgB, csgA, csgD and dsrB (Brown et al., 2001; Olsen et al., 1993b; Sledjeski et al., 1996). These data thus confirm and demonstrate, genome-wide, that low temperature serves as a primary environmental stimulus that causes the co-ordinated expression of a large set of RpoS-dependent genes and specifically defines those RpoS-controlled genes activated in response to this cue. At the same time, it is important to note that for the remaining 175 genes, it is unknown what thermoregulatory mechanism controls their increased expression at 23 °C.
Genes expressed at 23 °C overlap the cold-shock response
In mesophiles, the cold-shock response is characterized by the transient, increased production of cold-shock proteins after a temperature decrease (generally >10 °C) that subsequently facilitates adaptation to prolonged growth at low temperature (Phadtare et al., 2000). Of the cold-shock-inducible (Csp) proteins (CspA, CspB, CspG and CspI), cspI expression showed a 3.9-fold increase during growth at 23 °C compared to 37 °C. CspI, a cspA homologue and thought to act as a chaperone that denatures RNA for more efficient translation at low temperature, was shown previously to have increased transcription and mRNA stability upon a shift to 15 °C (Wang et al., 1999). Several other genes from our microarray overlapped with genes that show increased expression in two other microarray studies in which cells were exposed to cold-shock conditions, either transiently (cfa, otsA, otsB, poxB, dps, ycjK, ycgF, ycgZ, yceP/bssS, yedA) or after prolonged (ompC, wrbA) growth at 15–16 °C (Phadtare & Inouye, 2004; Polissi et al., 2003).
Of particular interest are the genes proven to be important for viability under cold-shock conditions. In our study, otsA and otsB, genes required for the synthesis of trehalose, an osmoprotectant which increases cell viability when cells undergo cold shock at 4 °C (Kandror et al., 2002), showed increased mRNA levels (2.9- and 3.5-fold, respectively) at 23 °C, similar to previous studies in which these genes were induced in an RpoS-dependent manner at 16 °C (Kandror et al., 2002). Our results show that cfa transcription is increased 4.7-fold at 23 °C compared to 37 °C. Cyclopropane fatty acyl phospholipid synthase (Cfa) modifies membrane phospholipids, converting the fatty acid moieties of these lipids from the unsaturated to the cyclopropane form. It is hypothesized that this modification of fatty acids by Cfa might decrease membrane fluidity to allow adaptation to stressful conditions, supported by the fact that cfa mutants have an increased sensitivity to freeze–thaw cycles (Grogan & Cronan, 1984, 1997; Zhao et al., 2003). proP and proV also demonstrated increased expression at 23 °C (3.0- and 2.3-fold, respectively) consistent with previous results implicating low temperature (10 °C) as an inducing signal for production of this osmoprotectant transporter (Rajkumari & Gowrishankar, 2001, 2002). Given that the microarray results described here represent cells adapted to 23 °C for approximately 10 generations, these data suggest that strategies used to protect the cell upon dramatic shifts in temperature are also likely to be valuable for long-term adaptation to growth at ambient temperature.
Low temperature increases expression of genes associated with biofilm development
Notably, several genes with increased expression at 23 °C in our microarrays are implicated in biofilm development (Table 2). Multiple studies have described a role for curli in adherence, important for biofilm formation (Cookson et al., 2002; Olsen et al., 1993a; Prigent-Combaret et al., 2001; Romling et al., 1998; Vidal et al., 1998). In our microarray, all of the curli genes show dramatic increased expression at 23 °C. YaiC, the homologue to Salmonella AdrA, increases cellulose biosynthesis involved in biofilm development in E. coli and Salmonella enterica serovar Typhimurium (Romling et al., 2000; Zogaj et al., 2001) and its mRNA levels were increased 4.4-fold in our microarray experiments. mlrA, which encodes a positive regulator of curli (csgD and csgBA) operons in avian-pathogenic E. coli (Brown et al., 2001) and of adrA expression in S. enterica serovar Typhimurium (Garcia et al., 2004), is increased 4.2-fold at low temperature in our microarray. bolA has been shown to be important for biofilm formation in E. coli MC1061 at 37 °C in minimal medium (Vieira et al., 2004) and its transcription is induced in response to a variety of stresses (acid, heat, osmotic) (Santos et al., 1999); our microarrays indicate that low temperature increases bolA expression 3.5-fold. YceP (BssS), implicated in biofilm formation and quorum sensing (Domka et al., 2006), is increased 2.8-fold at 23 °C in our study. NhaR activates the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamine (Goller et al., 2006) and its transcription is increased 2.5-fold at low temperature. Together, these data suggest that low temperature is an important environmental cue used to increase expression of several biofilm genes.
Both RpoS/DsrA-dependent and -independent thermoregulatory mechanisms mediate increased gene expression at 23 °C
We chose a set of genes associated with biofilm development and the cold-shock response to determine if their low-temperature transcription was fully RpoS- and DsrA-dependent under our growth conditions (M9 glycerol medium) or whether other response regulators might be involved in the low-temperature induction of these genes. Five genes associated with biofilm formation were tested – adrA/yaiC, csgA, mlrA, bolA and nhaR, whereas three genes that overlapped the cold-shock response were assessed – otsA, dps and ycgZ. Expression of yceP/bssS was also measured which has been shown to be associated with both biofilm development and cold shock. In addition, we also assessed the expression of three other genes for which there was no information on their function or regulation – ymdA, ymgB and yhiM – which showed high levels of transcription at 23 °C in the microarrays (13.4-, 9.5- and 7.6-fold, respectively).
All of the genes associated with biofilm development were dependent upon both RpoS and DsrA for transcription at 23 °C in M9 glycerol medium. Confirming the microarray results, all of the genes demonstrated increased mRNA levels at 23 °C compared to 37 °C in the wild-type strain (Table 3). In the rpoS : : Tn10 and dsrA : : cat mutant strains, transcription of all of the biofilm-associated genes at 23 °C was reduced to levels similar to those measured at 37 °C in the wild-type strain, demonstrating the induction at 23 °C is dependent upon RpoS and DsrA. Similarly, expression at 23 °C of the cold-shock genes (otsA, yceP/bssS) and genes of unknown function (ymdA and yhiM) was also fully dependent upon these two regulators (Table 3). The novel finding that ymdA and yhiM are within the RpoS regulon demonstrate that there may be additional targets of RpoS/DsrA within our list of genes whose increased expression at low temperature is dependent upon these regulators. These genes had not been previously identified as being RpoS-dependent, despite genome-wide comparisons under other environmental conditions (Weber et al., 2005).
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). For ycgZ, reduced mRNA levels were observed in the rpoS : : Tn10 mutant compared to the wild-type strain, indicating that maximal expression at 23 °C is an additive effect of RpoS-dependent and -independent mechanisms. Notably, this effect did not apply to the dsrA : : cat mutant where ycgZ expression levels were equivalent to those measured in the wild-type strain at 23 °C. dps expression at 23 °C was fully dependent upon RpoS, but only partially dependent upon DsrA as evidenced by an intermediate level of expression of this gene in the dsrA : : cat mutant at 23 °C.
The transcription of ymgB was fully independent of RpoS and DsrA, identifying this gene as one whose increased expression at low temperature is fully mediated by an alternative thermoregulatory mechanism (Table 3). High-level expression of ymgB at 23 °C is retained in the rpoS : : Tn10 and dsrA : : cat mutant strains, similar to or greater than levels expressed in the wild-type strain. It is currently unknown how ymgB expression is increased at low temperature. During cold shock, increased mRNA stability has been identified as a major factor in the increase of certain transcripts (reviewed by Gualerzi et al., 2003) and it is possible that this may play a role during prolonged growth at 23 °C. Alternatively, other thermoregulatory factors, either protein or RNA in nature, may be critical in directly activating transcription of this gene.
Interestingly, ymgB is located within a region of the genome that contains six genes that are temperature-regulated based on our results and two of the genes, ycgZ and ycgF, are expressed in cold-shocked cells (Polissi et al., 2003). ycgZ-ymgA-ymgB-ymgC are encoded on one strand on the genome, whereas ycgF-ycgE are located divergently on the opposite strand. Their localization suggests the possibility that these two sets of genes may be organized in operons. However, combining our studies and those of others (Weber et al., 2005), transcription of ycgZ and ymgA are impacted by RpoS whereas ymgB is fully independent of RpoS, arguing that these genes are driven by different promoters. Further studies are required to determine the transcriptional units for these genes, their function and relevance for growth at 23 °C.
The increased expression at 23 °C of ymgB, adrA and otsA is modulated by growth medium
To determine if the thermoregulatory response was retained in other growth media for both RpoS-dependent (otsA, adrA) and RpoS-independent genes (ymgB), the wild-type strain DL1504 was grown in either M9 glucose or LB at both 37 and 23 °C (Fig. 1). For all three genes, there is higher expression at 23 than at 37 °C in M9 glucose, indicating that a temperature differential is retained and identifying temperature as an important cue in regulating the expression of these genes. Glucose as a carbon source did not greatly alter transcription of any of the genes at 37 °C, but had differential effects on mRNA levels at 23 °C. otsA expression is increased 2.6-fold, whereas ymgB and adrA expression at 23 °C is decreased 1.4- and 3.5-fold, respectively, in M9 glucose compared to M9 glycerol. Thus, the temperature differential between expression at 37 and 23 °C in M9 glucose expands (otsA) or contracts (ymgB, adrA) for these genes compared to growth in M9 glycerol.
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). In contrast, for adrA and otsA, growth in LB decreased expression for both genes at 23 °C such that differential expression based on temperature is either decreased (otsA) or abrogated (adrA). Overall, temperature serves as an important cue in regulating gene expression in various media and is a dominant cue for regulating ymgB expression, whereas otsA and adrA clearly demonstrate how E. coli can integrate nutritional cues that modify the thermoregulatory response.
A shift to low temperature rapidly increases mRNA levels of temperature-regulated genes
To test how quickly cells respond to low temperature, an exponentially growing 37 °C culture was shifted to 23 °C and gene expression was measured at times after the shift. For the RpoS-dependent gene otsA, mRNA levels increased 7.7-fold by 1 h and peaked at 2 h after the shift with levels 21.2-fold higher than the initial amount at 37 °C (Fig. 2). Levels of otsA mRNA subsequently decreased by 5 h and reached levels at 11 h that approximate the steady-state levels measured by qRT-PCR (Table 3). For the RpoS-independent gene ymgB, mRNA levels showed a similar rapid increase in response to the shift in low temperature, peaking at 2 h with levels 86.9-fold greater than the initial 37 °C culture, but subsequently decreasing by 11 h to levels approximately twofold greater than that measured at steady state (Table 3). For both these genes, there is a relatively rapid increase in mRNA levels, followed by a dramatic drop at 5 h before expression levels stabilize at the later time points, suggesting that these early time points represent the time period during which transcription is being fine-tuned to bring target gene expression to steady-state levels.
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). The delayed timing of peak mRNA levels for adrA/yaiC may represent that it is an indirect, rather than direct target of RpoS. adrA/yaiC expression is stimulated by the transcriptional regulator CsgD (Brombacher et al., 2003) such that the delay may be attributed to time needed for production of this regulator whose expression itself is RpoS-dependent at 23 °C (Brown et al., 2001; Olsen et al., 1993b). Overall, these data signify that bacteria can quickly respond to changing temperature by regulating gene expression through both RpoS-dependent and RpoS-independent thermoregulatory mechanisms.
The rpoS : : Tn10 and dsrA : : cat mutations do not greatly alter growth at 23 °C or viability at 4 °C
Given the large number of RpoS-dependent genes expressed at 23 °C, particularly those related to cold shock, we hypothesized that rpoS : : Tn10 and dsrA : : cat mutant strains might be impaired for growth at 23 °C or long-term viability at 4 °C in comparison to the wild-type strain. To assess growth at 23 °C, a 37 °C culture in early exponential phase was used to initiate two cultures (37 and 23 °C) in M9 minimal glycerol medium and optical density readings were taken at time points thereafter. At 37 °C, the growth rates were 2.27±0.17, 1.74±0.10 and 2.04±0.03 h per generation, respectively, in the wild-type, rpoS : : Tn10 and dsrA : : cat strains, whereas at 23 °C the growth rates were 5.12±0.11, 5.00±0.37 and 4.85±0.07 h per generation, respectively. While the growth rates were significantly slowed at 23 °C compared to 37 °C for all strains, the generation times did not greatly change between the mutants and the wild-type strain at either temperature.
Similarly, the viability of the wild-type, rpoS : : Tn10 and dsrA : : cat strains upon a shift to 4 °C did not differ greatly. Cells were grown to exponential phase, plated on M9 minimal agar containing glycerol, and incubated for varying times at 4 °C before transfer for outgrowth at 23 °C. All the strains behaved similarly with little loss in viability for up to 4 days incubation at 4 °C (data not shown). However, fewer than 10 % of the cells from all strains were viable after 6 days of incubation at 4 °C. These results indicate that the rpoS : : Tn10 and dsrA : : cat mutant strains are not impaired in their response to cold-shock conditions compared to the wild-type strain.
It is a bit perplexing that an RpoS response is induced in response to growth at low temperature to direct the expression of 122 genes, yet cells deficient in RpoS or DsrA do not appear to be significantly impacted with regard to low-temperature growth or viability at 4 °C. Unlike other stresses, the physiological role of RpoS in responding to non-optimal temperatures has not been fully elaborated. High temperature growth increases the expression of RpoS, primarily through decreased turnover (Muffler et al., 1997), and there is decreased viability of rpoS mutant strains at elevated temperatures (48–55 °C) in E. coli K-12 (Berney et al., 2006; Lange & Hengge-Aronis, 1991) and O157:H7 (Cheville et al., 1996). Except for a mention by Sledjeski and colleagues of similar results to our own that rpoS and dsrA mutants did not impact growth rate (Sledjeski et al., 1996), we have not found any experimental evidence in E. coli that a loss of rpoS decreases cell viability at low temperature. It is possible that under our in vitro conditions, where nutrients and moisture are present during incubation at 23 or 4 °C, the effect of their loss is not extremely detrimental, but it might be under other conditions. Indeed, in S. enterica serovar Typhimurium, rpoS mutant cells suspended in 0.85 or 6 % NaCl and incubated at 4.5 °C demonstrated decreased survival in comparison to a wild-type strain with the difference in survival between strains most pronounced after 10 days incubation (McMeechan et al., 2007). Thus, a combination of other stress (e.g. nutrient starvation or hyperosmolarity) with low temperature may make RpoS more important for survival. However, the relevance of temperature alone to the decreased viability is a bit difficult to evaluate as decreased cell survival was also observed at 37 °C for the rpoS mutant strain.
An alternative hypothesis exists that may explain the lack of any difference in phenotype between the wild-type, rpoS and dsrA mutant strains at low temperature. Gene expression results presented here indicate there is at least one alternate thermoregulatory mechanism, independent of RpoS, that is responsible for increasing gene expression at 23 °C (e.g ycgZ and ymgB). While we cannot say if this represents single or multiple pathways, it could be that those genes critical for low-temperature growth utilize such a pathway, either alone or in addition to the RpoS pathway, that ensures their expression under changing temperature conditions. Such a system appears to exist in the thermal adaptation of E. coli shifted first to 42 °C prior to elevation to temperatures >50 °C (Lange & Hengge-Aronis, 1991). These cells do not demonstrate thermal sensitivity, even in the absence of RpoS, indicating there are alternative methods for adjusting to non-optimal higher temperatures (Lange & Hengge-Aronis, 1991). Also, the tolerance for a high level of rpoS mutants within E. coli populations (Ferenci, 2003) indicates that survival at low temperature is probably not mediated purely by the general stress response.
Low temperature increases biofilm formation and rpoS/dsrA mutations decrease biofilm formation at 23 °C
Overall, our microarray studies revealed that several biofilm-associated genes are expressed more highly at low temperature (Table 2) and our results show they are dependent upon RpoS and DsrA for transcription at 23 °C (Table 3). Thus, temperature, RpoS and DsrA were tested to determine their effect on biofilm development in M9 glycerol medium. We assessed biofilm formation at times where the planktonic growth in the well was similar at both temperatures such that any effect on biofilm formation could be primarily attributed to temperature and not to differences in growth. At the earlier time point (37 °C=7 h, 23 °C=10 h), planktonic growth was similar (Fig. 3a) and biofilm development was equivalent at both temperatures and in all strains, indicating that none of these factors (temperature, RpoS or DsrA) impacts biofilm formation at this point (Fig. 3b). However, at the later time point (37 °C=22.4 h, 23 °C=27 h), biofilm formation was a statistically significant 1.5-fold higher at 23 °C compared to 37 °C, indicating that low temperature favours biofilm formation. Biofilm formation in the rpoS : : Tn10 and dsrA : : cat mutants showed a statistically significant reduction, in which biofilm levels were only 62 and 65 %, respectively, of those observed in the wild-type strain. Since growth of the wild-type strain and rpoS : : Tn10 mutant strain at 23 °C was identical at the later time point, these data indicate that RpoS is needed to attain maximal biofilm formation at 23 °C. In the dsrA : : cat mutant, planktonic growth was slightly slower than for the wild-type and rpoS : : Tn10 strains and this may have contributed to decreased biofilm formation in this mutant. However, the requirement of RpoS for maximal biofilm formation at 23 °C and our data demonstrating that increased transcription of the biofilm-associated genes we tested at 23 °C is RpoS-dependent (Table 3) would suggest that the loss of DsrA may directly impact biofilm formation due to decreased levels of RpoS. In contrast, at 37 °C at the later time point, the rpoS : : Tn10 and dsrA : : cat mutants were similarly efficient at biofilm formation and both showed statistically significant increased biofilm formation (1.2- and 1.5-fold, respectively) in comparison to the wild-type strain at 37 °C (Fig. 3b). Thus, from our experiments, both the rpoS : : Tn10 and dsrA : : cat mutations are deleterious to biofilm formation at 23 °C in the later stages of development, but are advantageous at 37 °C.
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). In contrast, data from Adams & McLean (1999) suggest that an rpoS deletion is deleterious to biofilm formation. These authors do not state at what temperature they conducted their studies, but if they were performed at low temperature their results would be in concordance with ours, indicating that an rpoS mutant has a deleterious effect on biofilm formation at 23 °C.
Conclusion
Overall, the studies here demonstrate that temperature has a dramatic effect on gene expression, signifying that adaptation to low temperature requires a co-ordinated, multifunctional response. These studies implicate RpoS and DsrA in the co-ordinated expression of a large subset of genes at 23 °C and are linked to expression of both cold-shock and biofilm development genes at 23 °C. However, there are clearly an even greater number of genes with increased expression at 23 °C for which the thermoregulatory mechanism is unknown. Evidence provided in this study of an additional thermoregulatory mechanism(s) responsible for increased gene expression at 23 °C raises the possibility that independent mechanisms exist to ensure the proper adaptation to low-temperature growth that is important for the bacterium as it encounters changing environmental conditions.
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ACKNOWLEDGEMENTS |
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Edited by: S. C. Andrews
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Received 25 July 2007;
revised 19 October 2007;
accepted 24 October 2007.
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), ymgB (
) and adrA (
) gene expression in the wild-type strain DL1504 in M9 glycerol medium. Relative levels of expression were measured by qRT-PCR and are shown relative to the amount in the starting 37 °C culture at t=0 grown in M9 glycerol medium. A representative experiment is shown. Errors are expressed as ±1 SD from the mean based on three measurements at each time point.