Cell lysis directed by {sigma}E in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli

Md. Shahinur Kabir1, Daisuke Yamashita1, Satoshi Koyama1, Taku Oshima4, Ken Kurokawa4, Maki Maeda3, Ryouichi Tsunedomi1, Masayuki Murata1, Chieko Wada2,5, Hirotada Mori2,3 and Mamoru Yamada1

1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
2 CREST, Nara Institute of Science and Technology, Nara 630-01, Japan
3 R&E Center of Informatics, Nara Institute of Science and Technology, Nara 630-01, Japan
4 Graduate School of Information Science, Nara Institute of Science and Technology, Nara 630-01, Japan
5 Laboratory of Plasma Membrane and Nuclear Signalling, Graduate School of Biostudies, 1302, Kyoto University, Kyoto 606-8502, Japan

Correspondence
Mamoru Yamada
m-yamada{at}yamaguchi-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding {sigma}E exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a {sigma}E-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified {sigma}E regulon genes, suggesting that a large number of genes are under the control of {sigma}E in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.


Complete microarray datasets, in compliance with MIAME guidelines (http://www.mged.org/miame), are available at http://genome.naist.jp/array/ and http://genome.naist.jp/array/RPOE1_download.html (raw and processed data).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Escherichia coli undergoes a decrease in viable cell number in early stationary phase, decreasing by one to two orders of magnitude in the number of c.f.u. when grown in rich media, and then retaining the remaining level of c.f.u. for many days (Zambrano et al., 1993). The ssnA gene, under the negative control of {sigma}S, has been shown to enhance the decline in viable cells (Talukder et al., 1996; Yamada et al., 1999). In parallel with the decline, cells that are unable to form colonies appear to be led to a lysis process when the level of active {sigma}E is elevated, as in a mutant with a deficiency in anti-{sigma}E factor or in cells that harbour the rpoE gene cloned in a multicopy plasmid (Nitta et al., 2000). However, most of the molecular mechanisms underlying these phenomena have not been elucidated. Generally, in cells damaged irreversibly by lethal stresses, a variety of structural molecules seem to be degraded by several endogeneous enzymes, such as peptidoglycan hydrolases (autolysins), proteases, phospholipases, RNases and DNases, but the involvement of such enzymes in lysis has not been demonstrated.

{sigma}E, first identified as a transcription factor for the rpoH gene, which encodes a main heat-shock {sigma} factor (Erickson & Gross, 1989; Wang & Kaguni, 1989), is involved in the expression of several E. coli genes (De Las Peñas et al., 1997a; Rouviëre et al., 1995) whose products deal with unfolded periplasmic or membrane proteins caused by heat shock or environmental stresses (Missiakas et al., 1997; Raina et al., 1995; Rouviëre et al., 1995). {sigma}E has been demonstrated to be an essential sigma factor in the organism at both high and low temperatures (De Las Peñas et al., 1997b; Hiratsu et al., 1995; Lipinska et al., 1989). The accumulation of unfolded extracytoplasmic proteins triggers intracellular active {sigma}E molecules (Mecsas et al., 1993) via a unique mechanism of {sigma}E modulation, in which RseA, RseB and RseC, encoded by the rpoE–rseABC operon, may be involved (De Las Peñas et al., 1997a; Missiakas et al., 1997; Missiakas & Raina, 1997). RseA, an inner membrane protein, functions as an anti-{sigma}E factor by interaction with the {sigma}E molecule at its N-terminal cytoplasmic domain. RseB, a periplasmic protein, binds to the C-terminal periplasmic domain of RseA, and is thought to function as a sensor for unfolded proteins. RseC is an inner-membrane protein, and seems to be a component of a reducing system of the superoxide sensor SoxR (Koo et al., 2003). When unfolded proteins are accumulated in the periplasm in response to stresses such as high temperature or chemicals, RseB separates from the complex consisting of RseB, RseA and {sigma}E, releasing {sigma}E in an active form in the cytoplasm. The active {sigma}E then induces transcription from the rpoE P2 promoter to allow its own autoinduction and also expression of the {sigma}E regulon genes (Raina et al., 1995; Rouviëre et al., 1995). Recent reports have demonstrated another mechanism to elevate the number of active {sigma}E molecules. DegS serves as a periplasmic stress sensor that recognizes the improperly exposed C-terminal sequences of outer-membrane porins by interaction with its PDZ domain, and the activated DegS triggers a proteolysis cascade of the anti-{sigma}E protein, releasing {sigma}E to activate RNA polymerase transcription of its own regulon (Kanehara et al., 2002; Walsh et al., 2003; Young & Hartl, 2003). The control mechanism of {sigma}E appears to be much more complicated, however, because intriguingly both {sigma}E and anti-{sigma}E are under further regulation by protease and phosphorylation, respectively (Dartigalongue et al., 2001a; Klein et al., 2003). Additionally, the expression of the {sigma}E gene is markedly increased in early stationary phase under general growth conditions, and may thus play a crucial role in the phase following (Nitta et al., 2000). Several {sigma}E regulon genes, including htrA and fkpA, which encode a periplasmic serine protease (Lipinska et al., 1988, 1989; Strauch et al., 1989) and a periplasmic peptidyl-prolyl isomerase (Danese & Silhavy, 1997), respectively, have been well characterized (Ishihama, 1999; Missiakas & Raina, 1997). Recently, molecular genetic approaches to search for {sigma}E regulon genes have been performed (Dartigalongue et al., 2001b; Rezuchova et al., 2003), increasing the number to 58 genes.

In the present study, we demonstrated a direct involvement of the rpoE gene in cell lysis in early stationary phase by using a transient expression system. The effect of transient expression on genes from the whole genome in exponential and stationary phase cells was also examined by DNA microarray. It was found that 156 genes were significantly upregulated, including the previously reported 31 {sigma}E regulon genes (Dartigalongue et al., 2001b; De Las Peñas et al., 1997a; Erickson & Gross, 1989; Rezuchova et al., 2003; Rouviëre et al., 1995; Wang & Kaguni, 1989). In addition, 76 down-regulated genes were identified. The number of upregulated genes and their characterized/possible cellular location and operon organization suggest the existence of many {sigma}E regulon genes in E. coli. Moreover, we discuss the possible genes related to {sigma}E-directed cell lysis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo (Kyoto, Japan) and New England Biolabs. DNA sequencing kits were obtained from Applied Biosystems Japan. Oligonucleotide primers were synthesized by Proligo Japan K. K. (Tokyo, Japan). Other chemicals were all of analytical grade.

Bacterial strains, plasmids and culture media.
The E. coli K-12 derivatives and plasmids used in this study are shown in Table 1. A plasmid collection (archive clones from H. Mori) of genomic genes from E. coli W3110 was used for expression of clpX, ftsH, htrA and yebA, which are under the control of the lac promoter and cloned with the lacI gene. Cell culture was performed using modified Luria–Bertani (LB) medium [1 % (w/v) Bactotryptone, 0·5 % (w/v) yeast extract, 0·5 % (w/v) NaCl] (Miller, 1992) at 37 °C under aerobic conditions by reciprocal shaking. Antibiotics were added at the following final concentrations: 50 µg ampicillin ml–1, 10 µg chloramphenicol ml–1, 25 µg kanamycin ml–1. Pre-cultured cells were transferred into LB medium and diluted to a turbidity corresponding to OD600 0·1, and grown for appropriate times. The rpoE gene under the control of the araBAD promoter was induced by the addition of arabinose at a final concentration of 0·1 % (w/v). The genes under the control of the lac promoter were induced by the addition of 0·1 mM IPTG. Cell lysis was followed by monitoring culture turbidity or by SDS-PAGE of the medium fraction, as described by Nitta et al. (2000). All these experiments were performed at least three times independently.


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Table 1. Bacterial strains and plasmids

 
Construction of an expression plasmid of rpoE.
Conventional recombinant DNA techniques were applied (Izu et al., 1997; Sambrook et al., 1989). The DNA fragment bearing the rpoE gene (but lacking its own promoters) from W3110 was cloned after PCR amplification, as described previously (Yamada et al., 1993), using two primers, 5'-CCCGAATTCTGGAGTGGCGTTTCGAT-3' and 5'-CAAGTCGACGCCTAATACCCTTATCC-3' with EcoRI and SalI sites, respectively, at their 5' ends, and W3110 genomic DNA as template. An amplified 0·66 kb DNA fragment was digested with EcoRI and SalI, and inserted downstream of the araBAD promoter in pBAD24, generating pBADRPOE. The integrity of the cloned rpoE gene was confirmed by nucleotide sequencing (Sanger et al., 1977).

Disruption of genomic genes.
hlpA-, htrA-, mdoGH- and yebA-disrupted mutants of W3110 derivatives were constructed. The construction was performed by essentially the same procedure as described previously (Tsunedomi et al., 2003), in which the targeted gene or a set of its upstream and downstream DNA fragments was cloned into a plasmid, the cml cassette bearing the chloramphenicol resistance gene was inserted into the gene or between the upstream and downstream DNA fragments, and homologous recombination was allowed to proceed between the constructed cml-inserted DNA fragment and the genomic gene of W3110. The cloning of the targeted gene or a set of its upstream and downstream DNA fragments was carried out by amplification of the DNA fragments by PCR with the W3110 genomic DNA as template. The PCR primers used are listed in Table 2. In the case of hlpA, the 1·0 kb DNA fragment just upstream from the first codon of hlpA and the 1·0 kb DNA fragment just downstream from its termination codon were amplified with hlpA-up5' and hlpA-up3', and hlpA-dn5' and hlpA-dn3', respectively. In mdoGH, the 1·0 kb fragment just upstream from the first codon of mdoG and the 1·0 kb fragment just downstream from the termination codon of mdoH were amplified with mdoGH-up5' and mdoGH-up3' and mdoGH-dn5' and mdoGH-dn3', respectively. The 2 kb DNA fragments bearing htrA or yebA were amplified with htrA-5' and htrA-3' and yebA-5' and yebA-3', respectively. The clones of genes or DNA fragments were confirmed by nucleotide sequencing, and the gene disruption was checked by comparison of PCR fragments of the targeted gene from the genomic DNA of disrupted strains with that from W3110.


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Table 2. Primers used in this study

 
Preparation of RNA and DNA microarray.
W3110 cells harbouring pBADRPOE or pBAD24 were cultured in LB medium, arabinose was added at 6 h (corresponding to the middle of exponential phase) or 12 h (corresponding to early stationary phase), and cultivation was further continued for 1 h to induce the rpoE gene. From the induced cells, total RNA was immediately prepared according to the hot phenol method (Aiba et al., 1981). After phenol/chloroform treatment and ethanol precipitation, the resultant RNA (about 100 µg) was resuspended in 100 mM sodium acetate, pH 5·5, 50 mM MgSO4, and treated at 37 °C for 1 h with 10 units of RNase-free DNase (Takara Shuzo) in a final volume of 100 µl. RNA was then recovered after phenol/chloroform treatment and ethanol precipitation. DNA microarray preparation, fluorescently-labelled cDNA preparation, array hybridization, and data capture and analysis were performed as described previously (Oshima et al., 2002). We obtained at least two independent data per gene for each induction experiment. Spots with a significantly lower (<0·25; i.e. a negative fold difference) or higher (>2; i.e. a positive fold difference) fluorescence ratio of the {sigma}E-induced sample to the control sample were considered to represent real differences, as described previously (Oshima et al., 2002). Functional classification of genes was performed according to the EcoCyc (Karp et al., 2002) and KEGG (Kanehisa et al., 2002) databases. The function of genes and the cellular localization of their products was derived or inferred from the GenoBase, SWISS-PROT and NCBI databases. Complete microarray datasets, in compliance with MIAME guidelines (http://www.mged.org/miame), are available at http://genome.naist.jp/array/ and http://genome.naist.jp/array/RPOE1_download.html (raw and processed data).

RT-PCR analysis.
RT-PCR analysis was performed using an mRNA Selective RT-PCR kit (Takara Shuzo) to examine the expression of several genes, as described previously (Nitta et al., 2000). The primer sets used are listed in Table 2. Total RNA was prepared as described above. The reverse transcription reaction was carried out at 50 °C for 15 min with 0·1 µg of total RNA and each downstream primer, and subsequently PCR consisting of denaturing at 85 °C for 1 min, annealing at 45 °C for 1 min and extension at 72 °C for 1 min was carried out using the two primers for each gene. The PCR products after 15, 20, 25 and 30 cycles for each gene were analysed by 0·9 % (w/v) agarose gel electrophoresis and stained with ethidium bromide. As a control, 10 µg total RNA was run by 1·2 % agarose gel electrophoresis followed by staining with ethidium bromide, to make sure that equivalent amounts of RNAs were used between {sigma}E-induced and control samples. The relative amounts of RT-PCR products on the gel were compared by measuring band density after the colour of the image taken had been reversed by using a model GS-700 Imaging Densitometer (Bio-Rad). Linearity of the amplification was observed at least up to the thirtieth cycle. Under our conditions, the RNA-selective RT-PCR was able to specifically detect mRNA, because no band was observed when reverse transcriptase was omitted.

Proteome analysis by radical-free and highly reducing two-dimensional PAGE.
W3110 and WK3 cells were grown in 200 ml LB medium for 24 h and cell growth was stopped by the addition of TCA at a final concentration of 5 % (v/v). Cells and proteins in the medium were then recovered as a pellet by low-speed centrifugation and washed with ether. The pellet was suspended in 1·0 ml buffer 1 [100 mM ammonium acetate, 15 mM magnesium acetate, 20 mM Tris/HCl, pH 7·6, 6 mM {beta}-mercaptoethanol, 0·5 mM phenylmethylsulphonyl fluoride], sonicated for 15 min at 20 kHz with 50 % duty cycle (on for 7·5 s then off for 7·5 s) (Bioruptor UCD-200TM, Cosmo Bio) at 0 °C and then centrifuged for 10 min at 900 g. The supernatant was centrifuged for a further 20 min at 10 000 g. The pellet from the latter centrifugation was resuspended in 2 ml buffer 1, centrifuged for 20 min at 10 000 g and then homogenized in 0·5 ml buffer 1. This fraction was defined as crude debris. The supernatant after centrifugation for 20 min at 10 000 g was centrifuged further for 180 min at 100 000 g. The supernatant was defined as post-ribosomal supernatant, and the pellet was resuspended in 0·5 ml buffer 1 and centrifuged for 10 min at 17 000 g. The supernatant was defined as crude ribosome.

The post-ribosomal supernatant, crude debris and crude ribosome fractions were resuspended in a solution of 67 % acetic acid and 33 mM MgCl2 and centrifuged for 10 min at 10 000 g. The pellets were resuspended in 2 ml of the same buffer, and the centrifugation procedure was repeated. The two supernatants obtained after this were combined and desalted by Sephadex G-25 (Medium). The samples were then lyophilized. Lyophilized protein (~1–2 mg per gel) was analysed by radical-free and highly reducing two-dimensional PAGE, essentially as described previously (Wada, 1986), except that the volume of glacial acetic acid used in the sample charging buffer (50x) was 7·4 ml, not 74 ml, and a gel thickness of 2 mm was used to improve the resolution (see http://www.osaka-med.ac.jp/~yhide/index.htm). Other procedures, including detection of protein spots, blotting to PVDF, microsequencing of the N-terminal amino acid sequence and MALDI-TOF MS analyses were performed as described previously (Oshima et al., 2002). Proteins that were detected in increased quantities (ratio >1·3) in WK3 compared with the wild-type were considered to be significantly changed.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Comparison of protein expression between anti-{sigma}E-deficient and parental strains
When grown in LB medium, WK3, an anti-{sigma}E-deficient strain, showed a greatly decreased optical density and lysed in early stationary phase (Nitta et al., 2000). In order to identify proteins altered in expression in this phase, we compared the protein expression patterns of WK3 and its parental strain, W3110, by radical-free and highly reducing two-dimensional PAGE followed by MALDI-TOF MS analysis (Fig. 1). Proteins identified as relatively increased spots in WK3 were: an integration host factor {beta} subunit (IHF-{beta}); a periplasmic glucan biosynthesis protein, MdoG; a heat-shock protein protease, HtrA; peptidyl-prolyl cis-trans isomerase, SurA; a glutamine-binding protein, GlnH; a ribosomal protein, L19; glyceraldehyde-3-phosphate dehydrogenase, GapA; a histone-like protein, HlpA; and a yet-uncharacterized protein, YggN. Those identified as relatively decreased spots were the outer-membrane proteins OmpA and OmpW. Of the former, MdoG, HtrA, SurA, HlpA and YggN are encoded by previously identified {sigma}E regulon genes. The elevation of the expression of these proteins suggests that the expression of {sigma}E regulon genes is higher in WK3 than in the parental strain, as expected.



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Fig. 1. Comparison of proteins from WK3 (b, d) with those from the parental strain (a, c). WK3 and the parental strain were grown in LB for 24 h and total proteins were recovered as a pellet by the addition of TCA, as described in Methods. The pellet was then subjected to radical-free and highly reducing two-dimensional PAGE. (c) and (d) are high-resolution pictures of the enclosed regions of (a) and (b), respectively. Straight lines indicate proteins that were detected in increased quantities (ratio >1·3) in WK3 compared to the parental strain (a, c) and in the parental strain compared to WK3 (b, d). The two-dimensional experiments were repeated at least three times, and these patterns were confirmed to be reproducible. The data from one such representative experiment are shown.

 
The involvement of some of these proteins in cell lysis was examined by using gene-disrupted strains of WK3. However, none of the derivatives with the disrupted mutation of htrA, hlpA or mdoGH was found to suppress cell lysis. The disruption of surA was not performed, because the gene has been demonstrated to be essential for stationary phase survival (Tormo et al., 1990). Further examination by DNA microarray with a transient expression system of rpoE encoding {sigma}E was thus performed as described below.

Effect of transient rpoE expression on cell growth
In order to examine whether {sigma}E is directly involved in cell lysis, we constructed an inducible expression plasmid, pBADRPOE, in which a DNA fragment bearing the rpoE gene, but not its promoter region, was placed downstream of the araBAD promoter. First, the condition for induction of rpoE from the promoter was established. W3110 cells harbouring the expression plasmid were grown, the rpoE gene was induced by the addition of arabinose, and its expression was evaluated by RT-PCR. rpoE expression reached a maximum around 1 h after the addition of arabinose, and 0·1 % arabinose was found to be sufficient for its induction. When rpoE induction was initiated at 12 h, in early stationary phase, the turbidity of the culture gradually decreased, as shown in Fig. 2. Cell lysis was then examined by SDS-PAGE, and a large accumulation of protein in the medium was found after 13 h induction, as previously observed in WK3 (Nitta et al., 2000). These results suggest that {sigma}E directly causes cell lysis in early stationary phase.



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Fig. 2. Effect of rpoE expression on cell culture turbidity. W3110 cells harbouring pBADRPOE ({bullet}) or the vector pBAD24 ({circ}) were grown in LB medium containing ampicillin, and arabinose was added at a final concentration of 0·1 % to induce rpoE expression at 6 h (a) or 12 h (b) (indicated by arrowheads). The experiments were repeated three times. Error bars represent standard deviation from the mean.

 
On the other hand, the induction initiated at 6 h, corresponding to the middle of exponential phase, caused no significant decrease in the turbidity of the culture immediately after the addition of arabinose, but further continuation of the culture caused a reduction of the turbidity following a transient increase in the late exponential phase. Interestingly, the turbidity after about 40 h became nearly the same as that at the time of induction at 12 h. Although the reduction ratio was slightly different between the two conditions, both patterns of change in turbidity in the early stationary phase were similar to that observed in WK3 (Nitta et al., 2000). Such a phase-specific reduction in turbidity may be due to the lysis of viable but unculturable cells, formed at the beginning of stationary phase (Nitta et al., 2000). The slight difference in the reduction ratio between 6 h and 12 h induction would be due to the timing of the initiation of cell lysis: cell lysis for 6 h induction started earlier than that for 12 h induction. Therefore, the {sigma}E-directed decline in culture turbidity may occur specifically in the early stationary phase.

Change in expression of genomic genes following induction of rpoE
The results presented above prompted us to examine the change in global gene expression caused by induction of rpoE expression using a DNA microarray that included most E. coli genes. For probe preparation, on the basis of the results described above, W3110 cells harbouring pBADRPOE or pBAD24 were grown for 6 h (exponential phase) or for 12 h (early stationary phase) and further grown for 1 h in the presence of 0·1 % (w/v) arabinose. From the 1 h-induced cells, total RNA was isolated. The RNA for control probes was isolated from cells harbouring the vector pBAD24 that had been grown in parallel under the same conditions. The genes whose expression was significantly affected under the {sigma}E-induced condition were defined as those that exhibited a fluorescence intensity more than two times greater than that of the control or less than 25 % of that of the control, and such genes were classified into upregulated genes and down-regulated genes, respectively, as shown in Table 3.


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Table 3. Genes significantly upregulated and down-regulated in DNA microarray analysis

 
Among the 4000 genes tested, over 230 genes, including many functionally yet-uncharacterized (y) genes, showed significant alteration in their expression levels. There were 115 and 83 upregulated genes in cells at the exponential and early stationary phases, respectively, and 44 and 41 down-regulated genes, respectively (Table 3). In both phases, most {sigma}E regulon genes previously identified (bacA, cutC, dapA, ddg, dnaG, dsbC, ecfG (ygiM), fabZ, fkpA, fusA, hlpA, htrA, imp (ostA), ksgA, lpxA, mdoG, nlpB, psd, rpoD, rpoH, rseA, rseB, sbmA, smpA, surA, uspD (yiiT), xerD, yaiW, yfeY and ypfJ) (Dartigalongue et al., 2001b; Ishihama, 1999; Lipinska et al., 1989; Rezuchova et al., 2003) were also upregulated (Table 4), indicating that the {sigma}E regulon genes were induced under the conditions applied. Among the genes of proteins detected by proteome analysis shown in Fig. 1, htrA, mdoG, rplS and surA were detected as upregulated, and ompW as down-regulated in stationary phase. Additionally, down-regulation of the ompN gene was consistent with the finding, which was observed by one-dimensional SDS-PAGE (data not shown), that OmpN and/or OmpC was greatly reduced in WK3 compared to the wild-type strain.


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Table 4. Genes upregulated in DNA microarray, and operon organization of possible {sigma}E regulon

In exponential phase (Exp.) or early stationary phase (Sta.), expression ratios of more than twofold are shown. Location abbreviations: OM, outer-membrane proteins; IM, inner-membrane proteins; PP, periplasmic proteins; CP, cytoplasmic proteins; MA, membrane-associated proteins; TM, having transmembrane domain. The presence of a signal sequence for secretion is indicated by ‘+’. The categories in the Group column are the same as those shown in Table 3. Possible operon structures are shown according to expression pattern, gene arrangement on the genome, transcription direction, and previous reports. Underlining and asterisks indicate previously characterized rpoE regulon genes and their previously determined promoter sites, respectively. Alternative names are shown in parentheses.

 
Of the significantly upregulated genes, a number of genes encoded proteins related to membrane activities, the cell envelope (csgA ddg, flu, lpp, lpxA, lpxB, nlpB, ompA, pal and smpA), cellular processes (cutC, dsbC, fkpA, ftsH, ftsZ, hscB, lepB, mdoG, secM, secY and ygiS), fatty acid/phospholipid metabolism (accB, plsB and psd), and transport/binding (dcuD, focA, frwC, kdpA, kdpF, gatC, pstS, ptsN, ptsO, yeeF and znuA) were identified, as shown in Table 3. Many of these products function as extracytoplasmic proteins, as for known {sigma}E regulon genes. Other functional categories included genes for chaperones (surA and cspH) and tolerance proteins (imp and uspD).

The expression of representative genes upregulated in the exponential or early stationary phases was further analysed by RT-PCR (Table 5) with RNAs prepared by the same procedure as for the DNA microarray experiments. The ratio of the RT-PCR product from the arabinose-induced cells harbouring pBADRPOE to that from arabinose-induced cells harbouring pBAD24 was compared with the data of the DNA microarray shown in Table 4, and this indicated that the two sets of results were essentially consistent with each other.


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Table 5. Effects of rpoE induction on expression of genomic genes

The ratio was calculated by comparing the amount of the RT-PCR products obtained from cells harbouring pBADRPOE with that from cells harbouring the vector pBAD24, as described in Methods. The reported values are the mean of at least two independent experiments.

 
Possible {sigma}E regulon genes
It is possible that the upregulated genes found in this study are {sigma}E regulon genes. Indeed, out of 58 known {sigma}E regulon genes, 31 genes including rpoE were identified in this experiment, as described above. Since most of the known {sigma}E regulon genes encode extracytoplasmic proteins, characteristics of membrane-spanning segments and signal sequences for membrane proteins and secreted proteins, respectively, were searched in databases and classified as inner-membrane proteins (IM), outer-membrane proteins (OM) and periplasmic proteins (PP), in addition to membrane-associated proteins (MA) and cytoplasmic proteins (CP) (Table 4). The characteristics of the products of yaiW and yhjX were predicted according to their primary sequences deposited in databases. Out of 156 genes upregulated, 12, 14, 7 and 13 seem to encode PP, IM, OM and TM proteins, respectively. Additionally, nine proteins possess only the characteristic of an N-terminal signal sequence for secretion. Therefore, 44 % of the upregulated genes whose cellular locations had been determined, or were predicted, may encode extracytoplasmic proteins. The remaining genes are CP, MA and undefined proteins without a signal sequence or transmembrane segment(s).

Previously identified {sigma}E regulon genes that are underlined in Table 4 were found to be induced between 2·8- and 54-fold in exponential phase and to show between 2·7- and 27-fold upregulation in early stationary phase, respectively. The induction ratios of other upregulated genes in Table 4 were in the same range (except plsB) to those of the known {sigma}E regulon genes, suggesting again the possibility that they are under the control of {sigma}E. Altogether, most genes shown in Table 4 could be controlled by {sigma}E, although it cannot be ruled out that some genes are indirectly induced by alteration in the expression of {sigma}E regulon genes under the conditions tested. Further detailed experiments for each gene will be required.

Possible operon organizations of the upregulated genes were predicted on the basis of the gene arrangement, transcription direction and coordinated induction of expression with neighbouring genes in the genome (Table 4). We assumed that genes in a polycistronic operon would show a similar induction level, or some polarity in expression. As a result, 109 operon organizations, including previously demonstrated operons (underlined), of the {sigma}E regulon were predicted, in which there are 25 polycistronic and 84 monocistronic operons.

Genes encoding porins and transporters, and effect of cations on cell growth of WK3
Several genes encoding porins, nmpC, ompF, ompN, ompW and phoE (ompE), were repressed when rpoE was induced (Table 3). Of these, nmpC, ompN and ompW were down-regulated in both phases. Since some of these porin proteins may play a role similar to OmpC, which is required for the maintenance of the intracellular cation level, especially for stabilization of the ribosome complex (Apirakaramwong et al., 1998; Raj et al., 2002), the effect of Mg2+ on the growth of WK3 was investigated (Fig. 3). Supplementation with 20 mM Mg2+ increased the cell turbidity of WK3 without a significant change in the number of c.f.u. Therefore, the reduction of some porin proteins that may contribute to the uptake of Mg2+ into cells may be one of the causes of {sigma}E-directed cell lysis.



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Fig. 3. Effect of magnesium on cell turbidity and c.f.u. of W3110 ({blacksquare}) and WK3 ({circ}, {triangleup}, {square}). Cells were grown in LB medium for 12 h, and MgCl2 was then added (indicated by arrowheads) at a final concentration of 0 mM ({blacksquare}, {circ}), 2 mM ({triangleup}) or 20 mM ({square}). OD600 (a) and c.f.u. (b) were determined at the times indicated. The experiments were repeated three times. Error bars represent standard deviation from the mean.

 
The effects of Cu2+ and Zn2+ were also examined, because cutC, encoding a copper homeostasis protein, and znuA, encoding a high-affinity zinc uptake protein, were significantly expressed as rpoE was induced. On LB plates, the WK3 strain forms a very flat colony (presumably indicating lysis) instead of the typical raised colony of the parental strain. Interestingly, when grown in LB plates supplemented with 1 mM ZnCl2, 3 mM CuSO4 or 10 mM MgCl2, WK3 formed raised colonies. Such a change in colony morphology is probably to some extent due to the suppression of cell lysis.

Genes encoding proteins that cause degradation of cellular structural molecules
The degradation of cells that are irreversibly damaged occurs as a variety of structural molecules are exposed to the activated cellular degradation system, and by the action of endogenous degradative enzymes, including peptidoglycan hydrolases, proteases, phospholipases, RNases and DNases. Some genes for these enzymes may be induced along with rpoE expression. Several genes encoding such degradative enzymes were found among the upregulated genes: clpX, ftsH, htrA and yebA, encoding a protease or protease subunit; and recB, tatD and xseA, encoding a DNase or DNase subunit.

We investigated whether or not ClpX, FtsH, HtrA and YebA affected cell growth when overexpressed from plasmid clones (Fig. 4), even though yebA was not significantly induced in the early stationary phase. The induction of yebA and ftsH by the addition of IPTG resulted in a reduction in OD600. Protein accumulation in the medium, indicating cell lysis, was observed in cells harbouring the yebA or ftsH clone. Further experiments on yebA were performed with its disrupted WK3 derivative, but the mutation did not confer any significant protection against {sigma}E-directed cell lysis. Therefore, although YebA may play a role in the process of {sigma}E-directed cell lysis, other crucial factors may also participate. The effect of ftsH disruption was not tested, because this gene is understood to be essential for cell growth (Tomoyasu et al., 1993). It is thus possible that these proteases are involved in {sigma}E-directed cell lysis.



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Fig. 4. Effect of expression of the genes encoding proteases on cell culture turbidity. W3110 cells harbouring pCLPX (a), pHTRA (b), pFTSH (c) or pYEBA (d), bearing clpX, htrA, ftsH or yebA, respectively, were grown in LB medium containing chloramphenicol, and IPTG was added at 12 h (arrowhead). Closed circles ({bullet}) and open circles ({circ}) indicate conditions with and without 0·1 mM IPTG, respectively. The experiments were repeated three times and the patterns were confirmed to be reproducible. Data from one such representative experiment are shown.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
{sigma}E has been demonstrated to be involved in the response to extracytoplasmic stresses in E. coli. Here, we present evidence for an additional role that governs the lysis of unculturable cells in early stationary phase. Consistently, the {sigma}E gene becomes highly expressed at that time (Nitta et al., 2000). The experiments with a transient {sigma}E expression system applied in this study allow us to identify many candidate {sigma}E regulon genes in addition to previously characterized {sigma}E genes.

Considering {sigma}E-directed lysis specifically in the early stationary phase, it is possible that some genes induced in that phase are directly involved in lysis. Upregulated genes were apparently divided into three groups: genes induced specifically for the exponential phase, specifically for the early stationary phase, and induced in both phases (Tables 3 and 4). Genes or operons induced specifically for early stationary phase are apaG, cpxP, csgA, cysM, dcuD, dsbC, fis, frwC, ftsH, ftsZ, ksgA, lepB, lpxAB, malQ, oxyR, pabA, plsB, pstS, rluD, rplL, smpA, tatD, tktA, tldD, xseA, and several totally unknown y genes, and those that show an expression ratio in early stationary phase significantly higher (more than two times) than that in exponential phase are cspH, cutC, dsrB, ecfG, fusA, nlpB, pyrL, rpmC, rpoH, rseB, ybhP, ybhQ, ycbL, yfgK, yfgL, yijD and yqiK. Some of these genes or unknown genes could be involved in the process of cell lysis. However, the following possibility cannot be ruled out: gene(s) that are involved in lysis are expressed in exponential phase, but the activities of their gene product(s) responsible for cell lysis are somehow prevented by interaction with other proteins, by post-translational modification, or by rapid degradation. Additionally, there are other early stationary phase-specific upregulated genes that encode ribosomal proteins, regulatory proteins in the translation process, subunits for the secretion machinery, and proteins for cell division (Table 3).

In addition to upregulated genes, there were many down-regulated genes in both phases: 44 genes in exponential phase and 41 genes in early stationary phase (Table 3). ompN, ompW, tnaA, atoB, ada, rstB, nmpC and uspA showed a significant reduction in expression in both phases. Notably, Ada and UspA are known to be involved in the repair of damaged DNA and in protection against general stresses, including DNA damage (Gustavsson et al., 2002). On the other hand, the phase-specific reduction of grxB, encoding glutaredoxin 2, hupB, encoding DNA-binding protein HU–{beta}, and tpx, encoding thiol peroxidase, may affect cell survival, because their mutation raises sensitivity to oxidants, including hydrogen peroxide (Vlamis-Gardikas et al., 2002), reduces survival after prolonged starvation (Claret & Rouviere-Yaniv, 1997), and markedly slows growth in the presence of oxidative-stress-inducing reagents (Cha et al., 1996), respectively. The down-regulation of these genes may lead cells to be sensitive to oxidative stresses that would be increased after the late exponential phase, which may also be responsible for cell lysis. As mentioned above, the reduction in expression of porin genes may influence cell survival. Some mutations, such as the double disruption of ompC and rmf (Apirakaramwong et al., 1998; Samuel Raj et al., 2002) or of ompC and ompF (Nogami & Mizushima, 1983), have been shown to affect cell viability, presumably due to starvation of cations such as Mg2+. Thus, it is possible that the reduction of several Omp proteins is at least partially responsible for cell lysis.

In early stationary phase, the wild-type cells show a pronounced increase in expression of rpoE and protein accumulation in the medium, but to much lesser extent than WK3 cells or cells harbouring the rpoE clone (Nitta et al., 2000). {sigma}E-directed cell lysis thus appears to occur in the wild-type background. A large number of cells become incapable of forming colonies in the stationary phase (Nitta et al., 2000; Zambrano et al., 1993), and such cells may be gradually exposed to lysis during stationary phase. Enhanced expression of {sigma}E regulon genes may increase cell lysis under the conditions applied in this study. Notably, when rpoE expression was enhanced in exponential phase, cell density finally attained a peak similar to that attained by inducing rpoE expression in the early stationary phase (Fig. 2), suggesting that the {sigma}E regulon genes scarcely influence the number of viable cells, as demonstrated previously (Nitta et al., 2000). Cells from very late exponential phase to early stationary phase that are exposed to some stresses become viable but unculturable, which would result in lysis by the {sigma}E-directed system. {sigma}E-directed lysis of such unculturable cells may provide nutrients for the remaining cells and help to maintain a more-or-less constant cell number in stationary phase.

In WK3, a transposon insertion between the Shine–Dalgrano sequence and the initiation codon of rseA causes marked cell lysis in early stationary phase (Nitta et al., 2000). Considering the occurrence of rseA in an operon, rpoE–resABC, that is transcribed from two rpoE promoters, the insertion may reduce the expression not only of rseA but also of rseB and rseC. However, lowered expression of the latter two genes may not be responsible for the {sigma}E-directed cell lysis because introduction of the rseA gene alone can suppress the lysis of WK3 (data not shown). There are two possible explanations for this. First, the SoxR regulon, which has been demonstrated to be activated by a rseC-disrupted mutation (Koo et al., 2003), may not be related to this lysis. Second, RseA alone without RseB may function as an anti-{sigma}E, at least when it is overexpressed from the plasmid clone. It may thus be necessary to re-examine the physiological function of RseB.

During the analysis of {sigma}E-directed cell lysis, a large alteration in the expression of genomic genes was observed following {sigma}E induction. A number of possible {sigma}E regulon genes, including many yet-uncharacterized (y) genes, were identified. Although the molecular events in {sigma}E-directed cell lysis processes have not been defined, some clues to elucidate the mechanism were found. Further work, including increased expression or disruption of these genes, and isolation and characterization of suppressors of cell lysis, is required.


   ACKNOWLEDGEMENTS
 
We thank Drs O. Adachi, K. Matsushita and H. Toyama for their helpful discussion. We also thank K. Ito for assistance with research. This work was supported by a Grant-in-Aid for Basic Research from the Ministry of Education, Science and Culture of Japan (to M. Y.) and a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan (to T. O.)


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Received 2 March 2005; revised 26 May 2005; accepted 27 May 2005.



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