Figures
Abstract
The molecular mechanism supporting survival at a critical high temperature (CHT) in Escherichia coli was investigated. Genome-wide screening with a single-gene knockout library provided a list of genes indispensable for growth at 47°C, called thermotolerant genes. Genes for which expression was affected by exposure to CHT were identified by DNA chip analysis. Unexpectedly, the former contents did not overlap with the latter except for dnaJ and dnaK, indicating that a specific set of non-heat shock genes is required for the organism to survive under such a severe condition. More than half of the mutants of the thermotolerant genes were found to be sensitive to H2O2 at 30°C, suggesting that the mechanism of thermotolerance partially overlaps with that of oxidative stress resistance. Their encoded enzymes or proteins are related to outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control or cell division. DNA chip analyses of essential genes suggest that many of the genes encoding ribosomal proteins are down-regulated at CHT. Bioinformatics analysis and comparison with the genomic information of other microbes suggest that E. coli possesses several systems for survival at CHT. This analysis allows us to speculate that a lipopolysaccharide biosynthesis system for outer membrane organization and a sulfur-relay system for tRNA modification have been acquired by horizontal gene transfer.
Citation: Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, et al. (2011) Molecular Strategy for Survival at a Critical High Temperature in Eschierichia coli. PLoS ONE 6(6): e20063. https://doi.org/10.1371/journal.pone.0020063
Editor: Mick F. Tuite, University of Kent, United Kingdom
Received: January 6, 2011; Accepted: April 12, 2011; Published: June 10, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences and the Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology. This work was performed as collaborative research in the Asian Core Program between Yamaguchi University and Khon Kaen University, which was supported by the Scientific Cooperation Program agreed to by the Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT). These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Responses of Escherichia coli to high temperatures have been extensively investigated, though previous studies have mainly focused on the response to a temperature up-shift around 42°C, a response known as a heat shock response (HSR) to induce the expression of a set of proteins, heat-shock proteins (HSPs) [1]. The fact that many HSPs are conserved among species indicates that the actions of HSRs are the fundamentally and physiologically important mechanisms in living organisms [2], [3]. HSPs play crucial roles not only in the rescue or removal of proteins damaged by environmental stresses, including heat stress and salt stress, but also in the intrinsic folding of proteins under normal growth conditions [4].
It has been shown that 384 genes are up-regulated by short-time exposure to a temperature of 43°C as a heat shock in E. coli [5], and these genes may be directly or indirectly induced by the treatment. The directly induced genes encode HSPs, including the main cellular chaperone machineries of GroEL and DnaK, ATP-dependent proteases of Lon, HslUV, Clp and FtsH (HflB), periplasmic protease DegP, and other proteins involved in protein folding, refolding, quality control and degradation [6]. HSPs are under complex regulations and are divided into several regulatory groups by their major stimulons [7]. The control of their expression, however, is highly variable among organisms and even among various bacteria [8].
One of the control elements found in Gram-negative bacteria is a heat shock sigma factor that regulates transcription of the major HSPs. HSR in E. coli is generally mediated by alternative sigma factors, sigma 32 and sigma 24 [4], [7], [8]. Transcription of the rpoH gene for sigma 32 is induced at elevated temperature via the action of sigma 24 [7]. Sigma 24, which is inactive under non-stress conditions by interaction with anti-sigma factor, is activated by misfolding of outer membrane or periplasmic proteins and by stresses including heat shock [9]. Both sigma factors are further regulated at the translation level and or at the posttranslational level. The factor sigma 24 is in part regulated by a cognate small RNA, and sigma 32 synthesis is regulated by structural change of its own mRNA molecules serving as a cellular thermometer and its activity modulated by phosphorylation [10], [11]. Other microorganisms, on the other hand, appear to possess diverged regulatory mechanisms [12].
There is no information on the molecular mechanisms of response to and survival at a critical high temperature (CHT) in organisms, probably due to the limited experimental procedures. Developments of a single-gene knockout library and DNA chip analysis have encouraged us to perform a genome-wide investigation of responses in organisms under extreme conditions. Since several mesophilic bacteria including E. coli can grow and survive at high temperatures compared to other mesophilic bacteria, they are assumed to have acquired the potential for thermotolerance during their evolution. In this study, we utilized new procedures for the first time to obtain information on the molecular mechanisms related to thermotolerance in E. coli at CHT. Screening of thermosensitive mutants at CHT and informatics analysis of the corresponding genes revealed pathways or factors indispensable for survival at CHT. For essential genes, their possible involvement in the response to CHT was examined by DNA chip analysis. Based on the results, we propose novel molecular mechanisms for survival at CHT in E. coli.
Results and Discussion
Thermosensitive mutants and thermotolerant genes
In order to identify genes required for survival at CHT in E. coli, we screened for thermosensitive mutants from a single-gene knockout library [13], which had been constructed according to the one-step gene disruption method with an aph cassette [14] and for which each construct had been confirmed extensively [15]. In the disrupted gene of each mutant strain, the region between the 1st codon and the last 6 codons was displaced with the aph cassette, so that most of the coding region of the gene was deleted. Our experiments indicated that the parental strain used for construction of the disrupted library is able to grow at temperatures up to 47°C, this temperature thus being its CHT.
After three successive screening steps of the library, including 3,908 disrupted-mutant strains, 51 strains were found to be sensitive to CHT. Their growth curves at 37°C, 45°C and 46°C were then compared to those of the parental strain (Figure S1). The growth profiles suggest that most mutants selected are significantly sensitive to 46°C and some even to 45°C. Such a disrupted gene responsible for the thermosensitive phenotype was designated as a thermotolerant gene (Table 1 and Table 2).
The gene organization generated by construction of the disrupted mutants might give rise to a polar effect of the inserted aph gene on transcription of downstream genes that are intrinsically transcribed by read-through from the promoter or the region upstream of the disrupted gene. Such an organization was found in 42 of the 51 mutants. Sensitivity was not due to a polar effect in 29 of those 42 mutants because disruption of genes just downstream from the disrupted gene by the same method caused no thermosensitive phenotype. The remaining 13 mutants have either an essential gene or a thermotolerant gene as an immediate downstream gene (Figure S2). Their possible polar effects were thus tested by RT-PCR with total RNA prepared from cells exposed to a temperature of 37°C or 47°C (Figure S3). The results suggest that the transcription level of the immediate downstream gene in the mutant was almost the same as that in the parent in all cases except for the cases of mutants of aceF, tolQ, dnaK and rpsF. Most of these downstream genes would thus have their own promoters or the transcription level by read-through would be nearly the same as that of the aph promoter. However, the transcription levels of lpd, tolR and dnaJ located downstream of aceF, tolQ and dnaK, respectively, were increased and the level of rpsR located downstream of rpsF was decreased compared to those of the parental strain at both temperatures. Although the expressional alteration of the 4 genes was nearly the same at both temperatures, growth of the corresponding mutant strains at 37°C was not significantly changed from that of the parental strain. Taken together, the results suggest that the thermotolerant phenotype in the 51 mutants is due to disruption of the targeted gene and not due to a polar effect on its downstream genes. Out of the 51 thermotolerant genes, 8 genes, cydB, degP, dnaJ, dnaK, dnaQ, nlpI, rfaD and rfaC, had been reported as genes supporting growth at a high temperature [16]–[22], and thus we newly identified 43 thermotolerant genes in this organism.
Effects of supplements and oxidative stress on growth of thermosensitive mutant strains
Since LB was utilized as a medium for the screening of thermosensitive mutants, limitation of carbon source might cause sensitiveness to CHT. We thus examined the effect of glucose as a supplement for growth of the thermosensitive mutant strains (Table 1, Table 2 and Figure S4). We also tested the effect of MgCl2 because Mg2+ somehow protects against cell damage under stress conditions [23], [24]. The growth of 20 and 37 mutants was improved at CHT by the addition of 0.5% glucose and 20 mM MgCl2, respectively. The growth of sixteen mutant strains was improved by supplementation of not only glucose but also MgCl2.
Next, the effect of exogenous oxidative stress on the thermosensitive mutant strains was tested since a higher temperature causes more oxidative stress (Noor et al, 2009; unpublished data). We exposed thermosensitive mutant strains to 0.5 mM H2O2 in LB liquid medium at 30°C. Twenty-nine mutants were found to be sensitive to H2O2 (Table 1, Table 2 and Figure S4), corresponding to approximately 60% of the thermosensitive mutants. Moreover, out of the 10 thermosensitive mutants for which glucose and MgCl2 supplementation had no effect, 9 mutants showed sensitivity to H2O2. These results suggest that the mechanism of thermotolerance at CHT partially overlaps with that of oxidative stress resistance.
Bioinformatics analysis and classification of thermotolerant genes
To understand the molecular mechanism of E. coli survival at CHT, bioinformatics analysis with various public databases including the KEGG PATHWAY database was performed. Out of the 51 thermotolerant genes, 29 genes were successfully mapped on E. coli pathways in the KEGG PATHWAY database. Interestingly, many genes were found to be involved in the same metabolic pathway, suggesting that the organism possesses indispensable pathways at CHT. The remaining 19 genes except for 3 unknown genes were extensively analyzed by using the DDBJ or GenBank database. On the basis of results of these analyses and the effects of the supplements, the 51 thermotolerant genes were classified into 7 groups (Table 1 and Table 2).
Group A consists of genes concerned with energy metabolism for production of ATP. The gene products of aceE, aceF, lpd and ackA are mapped in the pyruvate metabolism pathway from pyruvate to acetyl CoA [25]–[28] and that of rpe is located in the pentose phosphate pathway. cydB and yhcB encode subunits of cytochrome d terminal oxidase, which generates the membrane potential responsible for ATP synthesis [16], [29]. lipA, which encodes LipA to produce lipoate required for pyruvate dehydrogenase reaction, also contributes to pyruvate metabolism [30]. Based on the results showing that disrupted mutations of these genes caused a thermosensitive phenotype, we assumed that the cells require more ATP at a higher temperature. This assumption was supported by the finding that the phenotype of most mutants in this group was partially suppressed by the addition of glucose (Table 1 and Figure S4).
Group B consists of genes related to biosynthesis of the cell wall or organization of the outer membrane. The products of gmhB, lpcA(gmhA), rfaC (waaC), rfaD (waaD/htrM), rfaE (gmhC), rfaF (waaF) and rfaG (waaG) were mapped into the lipopolysaccharide (LPS) biosynthesis pathway [17], [18], [31], [32]. The products of these genes are involved in synthesis of the heptose unit of ADP-L-glycero-D-manno-heptose from sedoheptulose-7phosphate or encode early heptosyl transferases for KDO-lipid A (rfaC and rfaF) and to further extend the inner core of LPS with glcosyltransferase (rfaG). ydcL, yfgL (bamB), ynbE, nlpI and ycdO encode peptidoglycan-associated outer membrane lipoproteins, and the products of pal, tolQ and tolR are components for a complex structure forming a biopolymer transporter [33], [34]. yciM encodes a protein possibly required for integrity of the outer membrane [35]. The thermosensitive phenotype caused by disrupted mutants of all of these genes was significantly suppressed by the addition of Mg2+ (Table 1 and Figure S4). Since Mg2+ is known to stabilize the outer membrane structure by binding extracellularly [36], it is assumed that YdcL, YfgL, YnbE, NlpI, YcdO, Pal, TolQ, TolR and YciM act as components or scaffold proteins of the membrane to maintain outer membrane integrity, especially at a high temperature. Similarly, our data suggest that Mg2+ is able to stabilize the outer membrane structure when the LPS biosynthesis pathway becomes defective.
Group C consists of dnaQ, holC, priA, ruvA and ruvC for DNA double-strand break repair (DSBR) [37]. DnaQ and HolC are epsilon and chi subunits, respectively, of DNA polymerase III [38], [39], which is required for homologous recombination in DSBR [19]. RuvA and RuvC act as DNA helicase and endonuclease, respectively [19], [40], before the replication restart in the DSBR process, and PriA functions as DNA helicase after the replication restart [41]. The requirement of DSBR for survival at CHT suggests that DNA molecules are subjected more to double-strand breaks at a higher temperature. Interestingly, mutants of all members in this group exhibited sensitivity to oxidative stress at 30°C. Therefore, it is thought that there is a strong connection between oxidative stress and DNA double-strand breaks.
Group D includes genes for tRNA modification. Products of iscS, yheL (tusB), yheM (tusC), yheN (tusD) and yhhP (tusA) have been demonstrated to compose the sulfur-relay system [42]–[44]. IscS is a widely distributed cysteine desulfurase that catalyzes desulfuration of L-cysteine by transfer of the sulfur to its active-site cysteine to form a persulfide group (-SSH), being responsible together with YheL, YheM, YheN and YhhP for biosynthesis of the 2-thio modification of 5-methylaminomethyl-2-thiouridine (mnm5s2U) [43] and five different thio modifications in bacterial tRNAs [45]. IscS also works as a general sulfur donor in various metabolic pathways [46] including biosynthesis of iron-sulfur (Fe-S) cluster [47], thiamine [48], nicotinic acid and branched-chain amino acids [49]. Additionally, miaA, trmU and truA in this group are involved in tRNA modification. The mutations of genes related to sulfer modification cause the phenotype of sensitivity to anti-oxidation stress [44]. Consistently, our study provided evidence that mutants of this group exhibited hypersensitivity to oxidative stress. YheL, YheM, YheN and YhhP, which mainly function in tRNA modification [45], are conserved in thermotolerant bacteria in mesophiles (see Table S3), whereas iscS, a general sulfur donor, is widely conserved in mesophiles.
These findings suggest that tRNA modifications presented here are indispensable for growth at CHT.
Group E genes encode chaperones and a protease and thus contribute to the cellular process of regulating heat shock response: dnaK and dnaJ encode a chaperone and co-chaperone, respectively, for maturation of protein folding or refolding of unfolded proteins [20], [50], and degP encodes a chaperone/serine protease located in the periplasm [21]. The indispensability of these genes at CHT suggests that DnaK/DnaJ play a crucial role in dealing with unfolded proteins caused by CHT and that DegP plays an important role in the removal of damaged proteins that have accumulated at such a temperature. rseA in this group encodes an anti-sigma factor to keep sigma 24 inactive under non-stress conditions. The thermosensitivity caused by rseA disrupted mutation suggests that fine tuning of the intracellular level of active sigma 24 that regulates expression of chaperone or protease genes is somehow crucial for adaptation to the CHT condition. Alternatively, the defective mutant of rseA increased sigma 24 activity, which in turn decreased the production of outer membrane proteins via MicA or RybB as a sigma 24 regulon gene [51], resulting in membrane unstability and thermosensitiveness at CHT.
Genes in group F belong to the translation control apparatus. S6 encoded by rpsF interacts with the central domain of 16S rRNA and has been demonstrated to play a regulatory rather than a structural role in the ribosome [52]. L36 encoded by rpmJ is a component of the 50S subunit of the ribosome, and its disruption decreases the expression of secY [53], which encodes a protein-conducting channel in the cytoplasmic membrane. DksA encoded by dksA functions as a negative regulator for rRNA genes [54]. Overexpression of DksA has been shown to be a suppressor for a dnaK deletion mutation [55] and ensures replication completion by removing transcription roadblocks [56]. SmpB encoded by smpB is a component of the trans-translation process and performs rescue of stalled ribosomes with its binding partner, transfer-messenger RNA [57]. These lines of evidence suggest that several constituents in translation pathways are crucial for survival at CHT.
Finally, genes in group G are related to cell division. A xerC-encoded protein is a site-specific recombinase [58] and is essential for conversion of chromosome dimers to monomers during cell division. envC encodes a component of the cell division machinery that is a direct regulator of the cell wall hydrolase responsible for cell separation that is required for cell division [59]. DedD encoded by dedD is a membrane-anchored periplasmic protein involved in septation [60] and has been shown to participate in cytokinesis [61].
The functions of the remaining genes, ybgH, yciM and yhhH, are unknown. Notably, the thermosensitiveness of their mutations was partially suppressed by the addition of Mg2+. It is thus likely that their gene products are related to cellular activities similar to those in group B, C or G.
Possible acquisition of some thermotolerant genes by horizontal gene transfer
Two groups for outer membrane integrity and tRNA modification are almost completely conserved in limited bacterial species with optimal growth at a relatively high temperature (Table S1). Of these group members, genes for the LPS biosynthesis pathway, some lipoproteins and the sulfur-relay system are distributed in very limited bacterial species including Enterobacteriaceae (Tables S2 and Table S3). The sulfur-relay system classified in tRNA modification has been demonstrated to modify a few nucleotides of tRNA molecules, contributing to stabilization of their structure, and to be required for survival at an extremely high temperature in Thermous thermophilus [62] and it is also conserved in Thermoanaerobacter tengcongensis (Table S3). The mature LPS biosynthesis pathway for assembly of the outer membrane consists of many enzyme reactions, which was found to be dispensable at a lower temperature. Interestingly, this pathway is mostly conserved in Thermodesulfovibrio yellowstonii and Thermanaerovibrio acidaminovorans (Table S2). Enzymes in the LPS biosynthesis and sulfur-relay system in E. coli share about 40% sequence identity and about 50% sequence similarity to the corresponding enzymes in thermophilic bacteria. E. coli and its closely related bacteria would thus have acquired these genes of the two groups presumably by horizontal gene transfer during their evolution. Since the other five groups are widely conserved not only in thermotolerant mesophilic bacteria but also in other mesophilic bacteria, they would be intrinsically present in E. coli. This is consistent with the conserved nature of essentiality of the lipid A part of LPS and essentiality of synthesis of lipid IVA but dispensability of enzymes involved in extension of Kdo2-lipid A by various glycosyltransferases. This draws support from Re (rfaC) mutants with only tetraacylated lipid A exhibiting a very narrow growth range with ability to grow only under slow growth conditions on minimal medium around 23°C [18], suggesting overall importance of outer membrane integrity at CHT.
Expressional change caused by heat shock at CHT
None of the thermotolerant genes identified in this study were found to encode HSPs previously identified in E. coli except for dnaJ, dnaK, degP and dnaQ. To examine whether the thermotolerant genes were up-regulated at CHT or not, we examined transient change in expression of the genomic genes at CHT by DNA chip analysis. The results showed that 42 genes and 111 genes were significantly up-regulated and down-regulated, respectively (Table S4). The up-regulated genes were classified mainly into genes involved in the cellular process, and the down-regulated genes were classified into genes involved in energy metabolism, transport/binding protein and translation. However, none of the thermotolerant genes including degP and dnaQ as a heat-shock gene were identified as up-regulated genes except for dnaJ and dnaK. Taken together with data shown above, it is possible that the chaperone systems except for DnaJ/DnaK and GroEL/GroES are not neccesarily involved in thermotolerant mechanisms acquired at CHT. Therefore, it is likely that most products of thermotolerant genes are not HSPs and that the organism possesses a specific set of genes required for survival at CHT.
It is possible that some of the essential genes are crucial for growth at CHT. Such genes, however, could not be examined in this analysis because no disrupted mutants for these genes are available other than the conditional mutants. We thus listed essential genes with significant fluctuation in expression at CHT (Table 3). groEL (groL) encoding HSP was up-regulated, indicating the possibility that the gene product contributes to survival at CHT. Consistently, it was reported that GroEL appears as a mediator of evolution of extremely heat-resistant E. coli cells [63]. On the other hand, 90% of the down-regulated genes were mapped into the translation pathway (Figure S5), encoding for components of ribosomal proteins. It is thus possible that down-regulation of ribosomal genes is one of the strategies for survival at CHT in E. coli. Noteworthily, Alix et al. reported that ribosome biogenesis in E. coli is high temperature-sensitive and DnaK-dependant and predicted that high temperature causes a severe limitation in DnaK/DnaJ to hamper ribosome assembly because heat-induced misfolded proteins would titrate out all the free DnaK/DnaJ [64], [65].
Further consideration on mechanisms for survival at CHT
Two groups of DNA double-strand repair and chaperone/proteinase genes may contribute to endurance against oxidative stress in addition to CHT. Evidence that a higher temperature results in accumulation of more oxidative stress [24] and the finding that mutants of all members in both groups exhibited sensitivity to oxidative stress allow us to speculate that oxidative stress is a main cause of DNA double-strand breaks and of damage to proteins at CHT. Interestingly, oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae [66], which is supported by the findings that overexpression of catalase and superoxide dismutase genes could increase the degree of thermotolerance and that the thermotolerance is increased under anaerobic conditions. We thus assume that CHT somehow causes intracellular oxidative stress to elicit harmful effects on cells as a secondary stress.
Significant suppression of the thermosensitive phenotype by a defect in the group of energy metabolism (Group A) by the addition of glucose suggests the limitation of energy level at CHT in the organism. The limitation seems to be resolved by alternative pathways that may generate ATP by glucose assimilation. The requirement of ATP at CHT may be consistent with expression of ribosomal genes. Many genes for ribosomal proteins were found to be down-regulated by exposure to CHT, and the disrupted mutant of dksA that encodes a negative regulator for rRNA genes became thermosensitive to CHT. These findings and evidence that translation as a ribosomal activity utilizes much energy, up to about 90% of energy consumed in cells [67], suggest that cells manage to reduce energy consumption under a severe condition at CHT. Such saved energy would be utilized for other crucial activities such as repair or degradation of damaged DNA or protein molecules. A smooth translational process at CHT might also save energy, for which S6 and L36 of ribosomal proteins in addition to SmpB may have important functions.
Several strategies for E. coli to survive at CHT were discovered. Most of them may also be responsible for other stresses and are conserved even in mesophilic bacteria. Early glycosyltransferases for LPS core biosynthesis for proper outer membrane assembly and permeability barrier function and the sulfur-relay system for tRNA modification might have been acquired for the organism to perform a main task to survive at CHT. Considering the genetic conversion of non-thermotolerant to thermotolerant bacteria, the two strategies might be applicable.
Materials and Methods
Materials
Oligonucleotide primers for polymerase chain reaction (PCR) were purchased from FASMAC Co, Ltd (Atsugi, Japan). Other chemicals were all of analytical grade.
Bacterial strains and growth conditions
Strains used in this study were derivatives of E. coli K-12. W3110 (IN (rrnD-rrnE), rph-1), BW25113 (rrnB3, Δ(lacZ)4787, hsdR514, Δ(araBAD)567, Δ(rhaBAD)568, rph-1) [14] and mutants of BW25113 in the Keio collection as a single-gene knockout library [13] were grown on plates or in liquid of modified Luria-Bertani (LB) medium (1% Bactotryptone, 0.5% yeast extract, and 0.5% NaCl) at 37°C, 45°C or 47°C for appropriate times.
Screening of thermosensitive mutants
The Keio collection consisting of 3,908 mutant strains was used for screening. In the 1st screening, mutant strains were grown on LB plates at 30°C overnight. A colony of each strain was patched on LB plates and incubated at 47°C for 48 h to find sensitive strains. The sensitive strains were subjected to the 2nd screening of spotting tests on plates. Cells were cultured in LB medium for 18 h and then diluted with LB medium to adjust turbidity to OD600 of 0.5, 0.05 and 0.005. The diluted samples (10 µl) were spotted on LB plates and incubated at 47°C for 48 h. The thermosensitive strains selected by the 2nd screening were subjected to the 3rd screening in liquid culture. After 8-h preculture, cells were diluted to a turbidity corresponding to OD600 of 0.1 and inoculated into LB medium at the final OD600 of 0.001. Samples were then incubated at 47°C for 18 h under a shaking condition. Thermosensitivity was determined by measuring OD600. Thermosensitive strains were defined to be <0.1 at OD600. The experiments were performed three times, and the results were confirmed to be reproducible.
Effects of glucose and MgCl2 and sensitivity to H2O2
To examine effects of supplements, glucose (0.5% (w/v)) or MgCl2 (20 mM) was added to the LB liquid culture. After 8-h preculture, cells were diluted to a turbidity corresponding to OD600 of 0.1 and inoculated into LB medium with or without the supplement at the final OD600 of 0.001. Samples were then incubated at 47°C for 18 h under a shaking condition. After 18 h, turbidity at OD600 was measured. To test the sensitivity to oxidative stress, H2O2 was added to the culture medium at the final concentration of 0.5 mM. After 8-h preculture, cells were diluted to a turbidity corresponding to OD600 of 0.1 and inoculated into LB medium with or without H2O2 at the final OD600 of 0.001. Samples were then incubated at 30°C for 8 h under a shaking condition. The experiments were performed three times, and the results were confirmed to be reproducible.
RT-PCR analysis
Cultures were grown in LB medium at 37°C until the exponential phase, and then the temperature was up-shifted to 47°C and incubation was continued for 8 min. Total RNA was immediately prepared from the heat-stressed cells by the hot phenol method [68]. RT-PCR analysis was performed using an mRNA-selective RT-PCR kit (TAKARA BIO Inc, Otsu, Japan) to examine the expression of immediate downstream genes of disrupted genes as described previously [69]. The primer set used for each gene is shown in Table S5. The RT reaction was carried out at 42°C for 15 min, 85°C for 1 min, 45°C for 1 min and extension at 72°C for 2 min using the two specific primers for each gene. After the completion of 15, 20, 25 and 30 cycles, the PCR products were analyzed by 0.9% agarose gel electrophoresis and stained with ethidium bromide. The relative amounts of RT-PCR products on the gel were compared by measuring the band density after the color of the image taken had been reversed using a model GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc, Tokyo, Japan) [70].
Bioinformatics and phylogenetic analyses
Bioinformatics analysis was mainly performed according to the instructions of the KEGG site (http://www.genome.jp/kegg/). Databases of DDBJ (http://www.ddbj.nig.ac.jp) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) were also used.
DNA chip analysis
W3110 cultures were grown in LB medium at 37°C until the exponential phase, and then the temperature was up-shifted to 47°C and incubation was continued for 8 min. A control culture was incubated in parallel at 37°C for 8 min. Total RNA was immediately prepared from the heat-stressed cells by the hot phenol method [69]. Preparation of cDNA, fragmentation and the end-labeling of DNA fragments were performed according to the instruction manual from Affymetrix. The ENZO Bioarray terminal labeling kit (Enzo Life Sciences, Inc, New York, USA) was used to end-label DNA fragments. DNA hybridization, data capture and analyses were performed as described in the protocol supplied by Affymetrix and GCOS software (Affymetrix, Inc, California, USA). Two independent experiments were performed and four data sets (two data sets at 37°C: 37°C-1 and 37°C-2, two data sets at 47°C: 47°C-1 and 47°C-2) per gene were obtained. The expression ratio used here indicates the average of the ratios obtained in the two independent experiments. Spots with a significantly lower (<0.50; i.e., a negative fold difference) or higher (2>; i.e., a positive fold difference) fluorescence ratio of the heated sample to the control sample were considered to represent a real significant difference. Physiological function and functional classification of the genes were derived from the Genobase database (http://ecoli.aist-nara.ac.jp/). Array data are accessible through ArrayExpress accession number E-MEXP-3191.
Supporting Information
Figure S1.
Growth of thermosensitive mutants in LB liquid culture at different temperatures. Each 51 thermosensitive mutant strain (opened symbols) and the parental strain, BW25113 (closed symbols), were grown in 30 ml LB medium at 37°C (circles), 45°C (squares), or 46°C (triangles). At the times indicated, turbidity at OD600 was measured. A, group A; B, group B; C, group C; D, group D; E, group E; F, group F; G, group G; H, others.
https://doi.org/10.1371/journal.pone.0020063.s001
(TIF)
Figure S2.
Gene organizations around genes having either an essential gene or a thermotolerant gene as a just downstream gene. Gene organizations around 13 thermotolerant genes that have either an essential gene or a thermotolerant gene as a just downstream gene are depicted. Black boxes represent identified 13 thermotolerant genes. Grey boxes represent essential or thermotolerant genes. The direction of boxes shows the direction of transcription.
https://doi.org/10.1371/journal.pone.0020063.s002
(TIF)
Figure S3.
Testing of possible polar effects by the aph insertion. Total RNA was prepared from cells cultured at 37°C (A, C) and 47°C (B, D) as described in Materials and Methods. RT-PCR was performed with primers specific for a just downstream gene of each thermotolerant gene to amplify about 500-bp DNA fragments. (A and B) After RT reaction, PCR was performed 15, 20, 25 and 30 cycles and the products were analyzed. (C and D) As a control, each total RNA (10 µg) was submitted to 1.2% agarose gel electrophoresis and staining with ethidium bromide.
https://doi.org/10.1371/journal.pone.0020063.s003
(TIF)
Figure S4.
Effects of addition of glucose and MgCl2 and sensitivity to H2O2. Thermosensitive mutant strains are shown by gene names. Growth conditions are described in Materials and Methods. Black and white columns represent turbidity under the conditions with or without supplements (0.5% glucose (A) or 20 mM MgCl2 (B)) or 0.5 mM H2O2 (C).
https://doi.org/10.1371/journal.pone.0020063.s004
(TIF)
Figure S5.
Down-regulated genes for ribosomal proteins. Systematic analysis of gene function was performed with a database of KEGG PATHWAY. Down-regulated genes for ribosomal proteins were mapped into 6 operons.
https://doi.org/10.1371/journal.pone.0020063.s005
(TIF)
Table S1.
Distribution of thermotolerant genes in various bacteria.
https://doi.org/10.1371/journal.pone.0020063.s006
(DOC)
Table S2.
Distribution of thermotolerant genes in group B in various bacteria.
https://doi.org/10.1371/journal.pone.0020063.s007
(DOC)
Table S3.
Distribution of thermotolerant genes in group D in various bacteria.
https://doi.org/10.1371/journal.pone.0020063.s008
(DOC)
Table S4.
Genes significantly up-regulated and down-regulated at CHT.
https://doi.org/10.1371/journal.pone.0020063.s009
(DOC)
Acknowledgments
We thank K. Matsushita and T. Yakushi for helpful discussion, and J. Makino and A. Ishii for assisting E. coli growth experiments. We also thank the National BioResource Project (NIG, Japan) for providing 3908 mutants from the KEIO Collection.
Author Contributions
Conceived and designed the experiments: MM HF SR NO MY. Performed the experiments: MM HF KN KC HN. Analyzed the data: MM HF TK TO MY. Wrote the paper: MM HF SR TO MY.
References
- 1.
Morimoto RI, Kroeger PE, Cotto JJ (1996) The transcriptional regulation of heat shock genes. a plethora of heat shock factors and regulatory conditions. In: Feige U, Morimoto RI, Yahara I, Polla BS, editors. The transcriptional regulation of heat shock genes: A plethora of heat shock factors and regulatory conditions. Basle: Birkhäuser-Verlag. pp. 139–163.
- 2. Boorstein WR, Ziegelhoffer T, Craig EA (1994) Molecular evolution of the HSP70 multigene family. J Mol Evol 38: 1–17.
- 3. Gupta RS (1995) Evolution of the chaperonin families (Hsp60, Hsp10, and Tcp-1) of proteins and the origin of eukaryotic cells. Mol Microbiol 15: 1–11.
- 4.
Yura T, Kanemori M, Morita MT (2000) The heat shock response: regulation and function. In: Storz G, Hennecke H, editors. Bacterial stress responses. Washington, D.C.: ASM Press. pp. 3–18.
- 5. Gunasekera TS, Gsonka LN, Pally O (2008) Genome-wide transcriptional responses of Escherichia coli K-12 to continuous osmotic and heat stresses. J Bacteriol 190: 3712–3720.
- 6. Narberhaus F, Kaser R, Nocker A, Hennecke H (1998) A novel DNA element that controls bacterial heat-shock gene expression. Mol Microbiol 28: 315–323.
- 7. Erickson JW, Gross CA (1989) Identification of the sigma E subunit of Escherichia coli RNA polymerase: A second alternate sigma factor involved in high-temperature gene expression. Genes Dev 3: 1462–1471.
- 8. Raina S, Missiakas D, Georgopoulos C (1995) The rpoE gene encoding the sigma E (sigma 24) heat-shock sigma factor of Escherichia coli. EMBO J 14: 1043–1055.
- 9. Alba BM, Leeds JA, Onufryk C, Lu CZ, Gross CA (2002) DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stress response. Genes Dev 16: 2156–2168.
- 10. Morita M, Kanemori M, Yanagi H, Yura T (1999) Heat-induced synthesis of sigma32 in Escherichia coli: structural and functional dissection of rpoH mRNA secondary structure. J Bacteriol 181: 401–410.
- 11. Klein G, Dartigalongue C, Raina S (2003) Phosphorylation-mediated regulation of heat shock response in Escherichia coli. Mol Microbiol 48: 269–285.
- 12. Helmann JD, Chamberlin MJ (1988) Structure and function of bacterial sigma factors. Annu Rev Biochem 57: 839–872.
- 13. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 1–8.
- 14. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97: 6640–6645.
- 15. Yamamoto N, Nakahigashi K, Nakamichi T, Yoshino M, Takai Y, et al. (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5: 335–337.
- 16. Wall D, Delaney JM, Fayet O, Lipinska B, Yamamoto T, et al. (1992) arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochromed operon. J Bacteriol 174: 6554–6562.
- 17. Raina S, Georgopoulos C (1991) The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene. Nucleic Acids Res 19: 3811–3819.
- 18. Klein G, Lindner B, Brabetz W, Brade H, Raina S (2009) Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response. J Biol Chem 284: 15369–15389.
- 19. Adams DE, West SC (1996) Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration. J Mol Biol 263: 582–596.
- 20. Tomoyasu T, Ogura T, Tatsuta T, Bukau B (1998) Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli. Mol Microbiol 30: 567–581.
- 21. Lipinska B, Zylicz M, Georgopoulos C (1990) The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. J Bacteriol 172: 1791–1797.
- 22. Ohara M, Wu HC, Sankaran K, Rick PD (1999) Identification and characterization of a new lipoprotein, NlpI, in Escherichia coli K-12. J Bacteriol 181: 4318–4325.
- 23. Kabir MS, Yamashita D, Koyama S, Oshima T, Kurokawa K, et al. (2005) Cell lysis directed by sigmaE in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli. Microbiology 151: 2721–2735.
- 24. Noor R, Murata M, Yamada M (2009) Oxidative stress as a trigger for growth phase-specific sigmaE-dependent cell lysis in Escherichia coli. J Mol Microbiol Biotechnol 17: 177–187.
- 25. Haydon DJ, Quail MA, Guest JR (1993) A mutation causing constitutive synthesis of the pyruvate dehydrogenase complex in Escherichia coli is located within the pdhR gene. FEBS Lett 336: 43–47.
- 26. Lyngstadaas A, Løbner-Olesen A, Boye E (1995) Characterization of three genes in the dam-containing operon of Escherichia coli. Mol Gen Genet 247: 546–554.
- 27. Cassey B, Guest JR, Attwood MM (1988) Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli. FEMS Microbiol Lett 1159: 325–329.
- 28. Barak R, Abouhamad WN, Eisenbach M (1998) Both acetate kinase and acetyl coenzyme A synthetase are involved in acetate-stimulated change in the direction of flagellar rotation in Escherichia coli. J Bacteriol 180: 985–988.
- 29. Mogi T, Mizuochi-Asai E, Endou S, Akimoto S, Nakamura H (2006) Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli. Biochim Biophys Acta 1757: 860–864.
- 30. Reed KE, Cronan JE Jr (1993) Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J Bacteriol 175: 1325–1336.
- 31. Kneidinger B, Marolda C, Graninger M, Zamyatina A, McArthur F, et al. (2002) Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli. J Bacteriol 184: 363–369.
- 32. Roncero C, Casadaban MJ (1992) Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus. J Bacteriol 174: 3250–3260.
- 33. Gerding MA, Ogata Y, Pecora ND, Niki H, de Boer PA (2007) The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli. Mol Microbiol 63: 1008–1025.
- 34. Kampfenkel K, Braun V (1993) Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment. J Bacteriol 75: 4485–4491.
- 35. Niba ET, Naka Y, Nagase M, Mori H, Kitakawa M (2007) A genome-wide approach to identify the genes involved in biofilm formation in E. coli. DNA Res 14: 237–246.
- 36. Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67: 593–656.
- 37. Motamedi MR, Szigety SK, Rosenberg SM (1999) Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo. Genes Dev13: 2889–2903.
- 38. Fijalkowska IJ, Dunn RL, Schaaper RM (1997) Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity. J Bacteriol 179: 7435–7445.
- 39. Xiao H, Dong Z, O'Donnell M (1993) DNA polymerase III accessory proteins. IV. Characterization of chi and psi. J Biol Chem 268: 11779–11784.
- 40. Bennett RJ, West SC (1996) Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange. Proc Natl Acad Sci USA 93: 12217–12222.
- 41. Al-Deib AA, Mahdi AA, Lloyd RG (1996) Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J Bacteriol 178: 6782–6789.
- 42. Rojas DM, Vásquez CC (2005) Sensitivity to potassium tellurite of Escherichia coli cells deficient in CSD, CsdB and IscS cysteine desulfurases. Res Microbiol 156: 465–471.
- 43. Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T (2006) Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol Cell 21: 97–108.
- 44. Dahl C, Schulte A, Stockdreher Y, Hong C, Grimm F, et al. (2008) Structural and molecular genetic insight into a widespread sulfur oxidation pathway. J Mol Biol 384: 1287–1300.
- 45. Lauhon CT (2002) Requirement for IscS in biosynthesis of all thio-nucleosides in Escherichia coli. J Bacteriol 184: 6820–6829.
- 46. Mihara H, Esaki N (2002) Bacterial cysteine desulfurases: their function and mechanisms. Appl Microbiol Biotechnol 60: 12–30.
- 47. Frazzon J, Dean DR (2003) Formation of iron-sulfur clusters in bacteria: an emerging field in bioinorganic chemistry. Curr Opin Chem Biol 7: 166–173.
- 48. Taylor SV, Kelleher NL, Kinsland C, Chiu HJ, Costello CA, et al. (1998) Thiamin biosynthesis in Escherichia coli. Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation. J Biol Chem 273: 16555–16560.
- 49. Lauhon CT, Kambampati R (2000) The iscS gene in Escherichia coli is required for the biothynthesis of 4-thiouridine, thiamine, and NAD. J Biol Chem 280: 26760–26769.
- 50. Ben-Zvi AP, Goloubinoff P (2001) Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones. J Struct Biol 135: 84–93.
- 51. Valentin-Hansen P, Johansen J, Rasmussen AA (2007) Small RNAs controlling outer membrane porins. Curr Opin Microbiol 10: 152–155.
- 52. Britton RA, Lupski JR (1997) Isolation and characterization of suppressors of two Escherichia coli dnaG mutations, dnaG2903 and parB. Genetics 145: 867–875.
- 53. Ikegami A, Nishiyama K, Matsuyama S, Tokuda H (2005) Disruption of rpmJ encoding ribosomal protein L36 decreases the expression of secY upstream of the spc operon and inhibits protein translocation in Escherichia coli. Biosci Biotechnol Biochem 69: 1595–1602.
- 54. Perron K, Comte R, van Delden C (2005) DksA represses ribosomal gene transcription in Pseudomonas aeruginosa by interacting with RNA polymerase on ribosomal promoters. Mol Microbiol 56: 1087–1102.
- 55. Kang PJ, Craig EA (1990) Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation. J Bacteriol 172: 2055–2064.
- 56. Tehranchi AK, Blankschien MD, Zhang Y, Halliday JA, Srivatsan A, et al. (2010) The transcription factor DksA prevents conflicts between DNA replication and transcription machinery. Cell 141: 595–605.
- 57. Watts T, Cazier D, Healey D, Buskirk A (2009) SmpB contributes to reading frame selection in the translation of transfer-messenger RNA. J Mol Biol 391: 275–281.
- 58. Bloor AE, Cranenburgh RM (2006) An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72: 2520–2525.
- 59. Uehara T, Parzych KR, Dinh T, Bernhardt TG (2010) Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis. EMBO J 29: 1412–1422.
- 60. Gerding MA, Liu B, Bendezú FO, Hale CA, Bernhardt TG, et al. (2009) Self-enhanced accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR domain (DamX, DedD, and RlpA) in Escherichia coli cell constriction. J Bacteriol 191: 7383–7401.
- 61. Arends SJ, Williams K, Scott RJ, Rolong S, Popham DL, et al. (2010) Discovery and characterization of three new Escherichia coli septal ring proteins that contain a SPOR domain: DamX, DedD, and RlpA. J Bacteriol 192: 242–255.
- 62. Shigi N, Sakaguchi Y, Asai S, Suzuki T, Watanabe K (2008) Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur-containing cofactors. EMBO J 7: 3267–3278.
- 63. Rudolph B, Gebendorfer KM, Buchner J, Winter J (2010) Evolution of Escherichia coli for growth at high temperatures. J Biol Chem 285: 19029–19034.
- 64. Al Refaii A, Alix JH (2009) Ribosome biogenesis is temperature-dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ. Mol Microbiol 71: 748–762.
- 65. René O, Alix JH (2010) Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine. Nucleic Acids Res 39: 1855–1867.
- 66. Davidson JF, Whyte B, Bissinger PH, Schiestl RH (1996) Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93: 5116–5121.
- 67.
Lehninger AL, Nelson DL, Cov MM (1993) Protein Metabolism: Principles of Biochemistry, Neal V(ed) pp892. New York: Worth Publisher, Inc.
- 68. Aiba H, Adhya S, de Cromburgghe B (1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256: 11905–11910.
- 69. Tsunedomi R, Izu H, Kawai T, Matsushita K, Ferenci T, et al. (2003) The activator of GntII genes for gluconate metabolism, GntH, exerts negative control of GntR-regulated GntI genes in Escherichia coli. J Bacteriol 185: 1783–1795.
- 70. Nitta T, Nagamitsu H, Murata M, Izu H, Yamada M (2000) Function of the SigmaE Regulon in dead cell lysis in stationary phase in Escherichia coli. J Bacteriol 182: 5231–5237.