HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Noor, R. Articles by Yamada, M. Search for Related Content PubMed Articles by Noor, R. Articles by Yamada, M.

¹ Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8505, Japan
² Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
³ Division of Medical and Biochemical Microbiology, Research Center Borstel, Parkallee 22, Borstel 23845, Germany

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
To understand the mechanism of ^E-dependent cell lysis, we examined the consequences of deletion derivatives of rpoE regulators rseA, rseB and rseC on ^E transcription, on levels of free versus membrane-bound ^E and on OMP-biogenesis limiting factor(s) that could impact cell lysis. RT-PCR showed that individual nonpolar rseA and rseB increased the rpoE expression to varying extents, with pronounced induction in rseA. Significantly the ratio of soluble (free) versus membrane-bound form of RpoE increased in rseA, however without increase of its total amount, unraveling furthermore complexity in RpoE regulation. Significant characteristics of cell lysis, accompanied by a severe reduction in the levels of periplasmic OMP-folding factor (PpiD), were observed in rseA. The cell-lysis phenotype of rseA was suppressed by either rseA or ppiD plasmids, but neither by rseB nor by rseC clones. However, the cell lysis of the wild-type strain was almost completely repressed not only by the rseA clone but also by the rseB clone, suggesting RseB might be limiting in vivo. Thus, increase in the ratio of free ^E in rseA mutants with a concomitant reduction in PpiD levels can account for ^E-dependent lysis in concert with a potential role of small RNAs on the lysis process.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
^E is a heat-shock sigma factor that was first discovered in Escherichia coli as a positive regulator for ^H, another heat-shock sigma factor (Erickson & Gross 1989; Wang & Kaguni 1989; Raina et al. 1995; Rouvière et al. 1995). rpoE, encoding ^E, is an essential gene required for growth at both high and low temperatures (Hiratsu et al. 1995; De Las Peñas et al. 1997a). The gene is located in the rpoE-rseABC operon with two promoters P1 and P2 located upstream of the rpoE ORF, which are recognized presumably by ^D, the main sigma factor for the exponential growth, and ^E, respectively (Raina et al. 1995; Rouvière et al. 1995). Such a promoter organization allows a positive auto-regulation of the operon and its sustained transcription under variety of growth conditions. ^E is involved in the cellular response to extracytoplasmic stresses and appears to express over 100 genes, including rpoH encoding ^H and rpoD encoding ^D genes (Missiakas & Raina 1997; Dartigalongue et al. 2001; Kabir et al. 2005). To perform the task, an ingenious regulatory system for ^E activity seems to have evolved. Most of the ^E molecules usually remain sequestered by the interaction with the inner membrane protein RseA (Missiakas et al. 1997; De Las Peñas et al. 1997b; Collinet et al. 2000).

With the current evidence, it is established that RseA primarily functions as an antisigma factor for RpoE by sequestering it to the inner membrane thereby preventing the formation of ^E-RNA polymerase core complex. RseB is known to interact with periplasmic C-terminal domain of RseA and this interaction is supposed to furthermore fine-tune the negative regulation by RseA antisigma factor (De Las Peñas et al. 1997b; Missiakas et al. 1997). In vitro, it has been shown that RseB inhibits proteolysis of RseA by DegS (Cezairliyan & Sauer 2006). In parallel, RseB also seems to be required for the function of essential RseP (EcfE) protease that acts as a site 2 specific protease, cleaving RseA in the inner membrane after DegS protease action (Grigorova et al. 2004). The extent to which RseB plays role in the induction of ^E and its physiological significance in controlling ^E function is not clear. This is not surprising given the lack of any described robust phenotype of rseB mutants. However, RseC may exert a subtle but positive modulating effect on ^E (Missiakas et al. 1997) or function in the reducing system of the superoxide sensor SoxR, which is involved in protection of cells against superoxide and nitric oxide (Koo et al. 2003).

The programmed cell death (PCD) is a mechanism for the removal of damaged or unnecessary cells. In E. coli, ^E-dependent cell lysis has been demonstrated (Kabir & Yamada 2005) to be akin to the addiction-module (toxin–antitoxin)-directed (Hazan et al. 2004) and mutagen-directed PCDs (Walker 1996). A large number of cells defective in colony forming ability, that is, viable but nonculturable (VBNC) cells (Nitta et al. 2000; Desnues et al. 2003; Nyström 2003; Cuny et al. 2005), which accumulate in the E. coli early stationary phase, have been shown to be significantly lysed in the rseA::Tn10Kan background with increased expression of the rpoE encoding ^E. However, culturable cells capable of forming colonies are not affected (Nitta et al. 2000). The expression of rpoE increases in the wild-type strain in the early stationary phase (Nitta et al. 2000; Costanzo & Ades 2006), even though its cell lysis occurs to a lower extent than that of the rseA mutant. The VBNC population at the stationary phase appears to contain damaged cells, since the lysis is enhanced in an rpoS background (Kabir et al. 2004b). The global gene expression along with the process toward the lysis has been analyzed (Kabir et al. 2004a, 2005). However, the molecular mechanism of ^E-dependent cell lysis has not been elucidated yet.

Majority of the genes belonging to the RpoE regulon, whose transcription is positively regulated by E^E polymerase, seem to be dedicated to function for maintaining the integrity of outer membrane (OM) or maturation of outer membrane proteins (OMPs) (Missiakas et al. 1996a; Dartigalongue et al. 2001; Kabir et al. 2005). This includes genes like surA, skp, htrA, ecfK (yaeT) and some of the genes involved in LPS biosynthesis and its translocation. Collectively, they perform important functions for assembly and maturation of OMPs. However, none of the tested regulon members were found directly to be involved in cell lysis (Kabir et al. 2005). Paradoxically, the synthesis of major OMPs is negatively regulated upon RpoE induction, which is ascribed to RpoE-dependent induction of synthesis of noncoding small RNAs, which in turn repress the OMP synthesis at post-transcriptional level, presumably to maintain a tight control of OMP levels (Valentin-Hansen et al. 2007).

Since ^E-dependent cell lysis is a highly reproducible and quantifiable phenotype associated with ^E induction, we examined roles of rseABC genes and any additional limiting factors in its control. The fine-tuned regulation of concentration of the active ^E in response to cellular damage levels might decide either repairing damaged proteins or killing cells. In this work, we first focused on constituents of the rpoE-rseABC operon and examined the effects of deletion mutants and plasmid clones of the individual gene on the damaged cell lysis, since their relative contributions to ^E-dependent cell lysis are not known at present. Next, we found the levels of PpiD are severely diminished in rseA mutants, and the ppiD gene, when expressed from a plasmid, can efficiently suppress the cell lysis of rseA mutants, suggesting that cell lysis in rseA mutants due to RpoE activation results from defects in maturation and synthesis of OMPs. Presented data suggest that RseA and PpiD are capable of repressing the ^E-dependent cell lysis, that RseB might be limiting in vivo and that RseC protects a cell lysis phenomenon distinct from the ^E-dependent cell lysis at 45 °C.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Effect of the deletion mutations of rseA, rseB and rseC on the ^E-dependent cell lysis

We previously demonstrated that ^E-dependent cell lysis phenotype is characterized by decrease in cell density without significant influence in colony forming unit (CFU) and by protein accumulation in the medium under growth conditions of 37 °C (Nitta et al. 2000; Kabir et al. 2005). Since ^E was first identified as a heat-shock sigma factor (Erickson & Gross 1989; Wang & Kaguni 1989; Raina et al. 1995; Rouvière et al. 1995), we examined whether the deletion mutants of rseA, rseB and rseC showed altered cell lysis phenotype at different temperatures (Fig. 1). At all temperatures tested, rseA showed a significant decrease in optical density at 600 nm (OD₆₀₀), but rseB and rseC showed nearly similar OD₆₀₀ as compared to that of the wild-type strain (Fig. 1A). The reduction in OD₆₀₀ was more at higher temperature in rseA, accompanied by decrease in CFU. However, at 45 °C, further reduction in the OD₆₀₀ of rseA bacteria stopped after 48 h of incubation, where cells might cease the ^E-dependent lysis and presumably became completely defective cells, which were indicated by a large reduction of CFU upon prolonged incubation (60 or 72 h) (Fig. 1B). Notably, rseA, as well as the other two mutants, showed nearly similar patterns of CFU curves to that of the wild-type strain (Fig. 1B).

View larger version (42K): [in this window] [in a new window]   Figure 1  Effects of the rseA, rseB or rseC mutation on cell turbidity, CFU and protein accumulation in medium at different temperatures. Wild-type (open circles), rseA (closed circles), rseB (open triangles) and rseC (closed triangles) strains were grown in LB medium at 30, 37, 42 and 45 °C with shaking. (A) Cell turbidity at 600 nm (OD600) and (B) CFU were measured at various times as indicated. The experiments were carried out three times. Error bars represent standard deviation ± mean. (C) Detection of cell lysis through protein accumulation in medium fractions. Aliquots were removed from the culture at 12, 36 and 60 h, and were fractionated as described in Experimental procedures. Proteins in medium fractions, corresponding to 0.6 mL culture, were separated by 12% SDS–PAGE and stained with Coomassie brilliant blue. M represents prestained protein markers. The experiments were carried out three times, and the patterns were shown to be reproducible. Data from one representative experiment are shown.

At 45 °C, protein accumulation in rseA increased at 12 h compared to those at other temperatures. Interestingly, at this temperature, the decrease in OD₆₀₀ and the increase in accumulation of proteins in rseA appeared to cease around 48 h (Fig. 1A,C), presumably due to the high temperature stress, which resulted in drastic reduction in CFU upon furthermore incubation and may hamper the ^E-dependent lysis. An increase in protein accumulation in rseB and rseC strains at 45 °C over other temperatures was observed after 36 h as compared to the wild-type strain. Therefore, lack of RseB and RseC appears to have a minor effect for the protein accumulation in the ^E-dependent cell lysis up to 42 °C but may prevent cell lysis at higher temperature such as 45 °C. However, the cell lysis phenotype at 45 °C in case of rseC might be ^E-independent (see below). As a test for cell lysis, we measured β-galactosidase activity using the wild-type, rseA, rseB and rseC strains. The β-galactosidase activity in the medium fractions of wild type, rseA, rseB and rseC were estimated around 19%, 85%, 23% and 16%, respectively, of the sum of that of medium and remaining fractions. The significant increase in β-galactosidase activity in rseA medium fraction indicates the accumulation of cytoplasmic proteins in medium due to cell lysis.

Complementation of the rseA phenotype by the rseA plasmid clone

Although the deleted regions in rseA, rseB and rseC encompassed from the second codon of each gene to the front of the Shine–Dalgarno sequence for the just downstream gene, we examined the complementary effect of the rseA, rseB or rseC plasmid clone on the rseA strain. Examination of the colony morphology on LB plates after 48 h incubation showed that the rseA strain formed flat and thin colonies at temperatures above 37 °C and slightly flat colonies at 30 °C, which seems to be consistent with the accumulated protein level in liquid medium. The formation of flat colonies seems to reflect the lysis of cells defective in colony formation. In contrast, both rseB and rseC showed round colonies, thus being indistinguishable from those of the parental strain. Introduction of the rseA plasmid clone, but not the rseB or rseC plasmid clone, changed the rseA strain to form round colonies similar to those of the wild-type strain. The effect of these plasmid clones on the OD₆₀₀ in growth or accumulation of proteins in the rseA strain was also tested. Only the rseA plasmid clone recovered the OD₆₀₀ (data not shown) and reduced protein accumulation (Fig. 2A) in medium to the level of the wild-type strain.

View larger version (47K): [in this window] [in a new window]   Figure 2  Effects of introduction of rseA, rseB or rseC plasmid clone on the cell lysis in rseA and the wild-type (WT) strains. (A) rseA/pBAD24 (empty vector), rseA/pBADRSEA, rseA/pBADRSEB and rseA/pBADRSEC cells, (B) W3110/pBAD24, W3110/pBADRSEA, W3110/pBADRSEB and W3110/pBADRSEC cells or (C) rseB/pBAD24, rseB /pBADRSEA cells were grown in LB medium at 37 °C with shaking. Aliquots were removed from the culture at 12, 36 and 60 h, and were fractionated as described in Experimental procedures. Proteins in medium fractions from 0.6 mL culture of rseA strain and from 12 mL culture of the wild-type or rseB strain were examined as in Fig. 1C. M represents prestained protein markers. The experiments were carried out three times, and the patterns were shown to be reproducible. Data from one representative experiment are shown.

Effect of rseA, rseB or rseC plasmid clones on the ^E-dependent cell lysis in the wild-type strain

As reported previously, the wild-type strain also showed accumulation of proteins in liquid medium in early stationary phase, consistent with increased expression of the rpoE gene in this phase (Nitta et al. 2000). If the ^E-dependent cell lysis was responsible for this protein accumulation, the control factors for the rpoE expression were assumed to repress the protein accumulation of the wild-type cells. The rseA, rseB and rseC plasmid clones were thus introduced into the wild-type strain and examined for protein accumulation in the culture medium. Samples of medium fractions of the wild type, equivalent to a 20-fold volume of that of the rseA mutant strain used in experiments for which results are shown in Figs 1(C) and 2(A), were applied on 12% SDS–PAGE. This analysis showed that not only rseA clone, but also rseB clone repressed the protein accumulation (Fig. 2B). The amount of proteins observed at every time of sampling shows this phenomenon of reduction in the accumulated proteins by introduction of either rseA or rseB plasmids (Fig. 2B). The expression of these genes from the plasmid clones was assumed to be very low because arabinose as an inducer was not added in the culture medium. Given the suppression observed by RseB plasmid in the wild type only but not in rseA, suggest that RseB-mediated suppression of cell lysis requires rseA wild-type copy. Consistent with these results, introduction of rseA plasmid clone in rseB also caused repression of cell lysis phenotype as was observed in the wild type (Fig. 2C). Taken together, it can be concluded that RseA suppresses the lysis in the wild-type strain independent of RseB. In contrast, RseC was found to slightly increase the protein accumulation in the wild type. This result is consistent with the minor protein bands in rseC compared to the wild-type or rseA or rseB strains at 37 °C and 42 °C as observed in Fig. 1(C). Therefore, these data suggest a minor positive role for RseC in the regulation of cell lysis control by rpoE-rseABC operon, supporting a similar previously proposed role for RpoE regulation (Missiakas et al. 1997).

Effect of rseA and rseB plasmid clones on the transcription from rpoEP2 promoter in rseA and the wild-type strain

As showed from data presented in Fig. 2, the suppression of lysis by RseB in the wild-type strain, but not in rseA, led us to assume that RseB function requires RseA to negatively regulate rpoE-rseABC operon. Therefore, the rpoE activity was assayed by measuring the activity of rpoE-dependent promoter fusions both in rseA null mutant and the wild-type strain, harboring either RseA or RseB plasmid clones. From the transcriptional assay using single-copy chromosomal rpoEP2 promoter fused with lacZ, in rseA null mutant containing either rseA or rseB plasmids showed that RseA severely repressed the rpoEP2 promoter activity, whereas the repression by RseB was insignificant in this mutant as compared to that in the empty vector (Fig. 3A). In contrast, the transcriptional assay in the wild-type strain showed that both RseA and RseB suppressed rpoEP2 promoter activity, although the magnitude of repression was higher with RseA than RseB (Fig. 3C). Taken together, with the results from Fig. 2, these data suggest that ^E response and consequent control of cell lysis by RseB acts primarily via RseA.

View larger version (15K): [in this window] [in a new window]   Figure 3  Effects of rseA and rseB plasmid clones on the expression of rpoEP2 promoter in (A) rseA and (C) wild-type (WT) strains. Empty vector (open circles), RseA plasmid clone (closed circles) or RseB plasmid clone (triangles) were electroporated to the cells carrying rpoEP2-lacZ promoter fusion on the genome. Selected transformants were grown at 37 °C, and samples taken at the times indicated were subjected to a β-galactosidase assay as described in Experimental procedures. The growth of rseA (B) and the wild type (D) was measured by the absorbance at 600 nm at specific time points used simultaneously for β-galactosidase measurements. The experiments were carried out on four independently obtained transformants and one representative data set has been shown.

To furthermore examine the effects of the three deletion mutations on the ^E-dependent cell lysis, the expression of rpoE was compared with the rseA, rseB, rseC and wild-type strains. Total RNAs from cells that were grown until early stationary phase at 37–45 °C were subjected to RT-PCR. The mRNA level of rpoE was quantified from the intensities of PCR products after agarose gel electrophoresis (Supporting Information/Supplementary Material Fig. S1). Considering the number of the PCR cycle, the rpoE expression in rseA was estimated to be 5- to 20-fold higher than that in the wild type at different temperatures. Approximately twofold higher expression of rpoE was observed in rseB, and no significant difference in rseC was found. These results furthermore establish that functional transcriptional activity is primarily enhanced in rseA and modestly in rseB.

Effect of the deletion mutations of rseA, rseB and rseC on the amounts of ^E and its association with inner membrane

Next, the expression level and cellular fractionation of ^E protein were compared with the three deletion mutants (Fig. 4A–C), since the extent of influence by defined individual nonpolar deletion mutants is not clear enough. The total cell extracts or fractionated samples from cells grown until early stationary phase, which was the same condition as for the study of the rpoE transcription, were subjected to Western blot analysis. As a control for fractionation of soluble and inner membrane proteins, we analyzed the corresponding samples for the presence of relatively abundant known cytosolic heat-shock protease subunit ClpY (Missiakas et al. 1996b). As can be seen, no ClpY could be detected in the inner membrane (IM) fractions of either wild type or those from rseA/B/C mutants (Fig. 4G), but was present only in the soluble fractions. Furthermore, PpiD served as a perfect marker for IM fractionation given its exclusive inner membrane localization due to its single N-terminal inner membrane anchor (Dartigalongue & Raina 1998) (see Fig. 5A). To ensure the authenticity of RpoE recognition, total cell extracts from rpoE were applied as additional control (Fig. 4A). The analyses of fractionation experiments showed that the total RpoE amounts in cell extracts did not increase in either rseA or rseB mutants, rather only the ratio between soluble versus IM-associated forms increased (Fig. 4A,B). However, RpoE amount in soluble fraction was found to be lower in rseA and rseB mutants than the wild type. Of significance, the rseA and the rseB mutants showed decrease in IM association of RpoE was much more pronounced in rseA (Fig. 4C). These results together with those of RT-PCR can explain constitutive induction of the rpoE regulon in rseA mutants with only a minor induction in rseB mutants. Thus, the key to induction of RpoE regulon seems to be extent of its decrease in IM association and increased ratio of soluble versus IM form, rather than overall amounts of RpoE. Interestingly, a fraction of RpoE was still found to be present in the IM of rseA (Fig. 4C). These results thus show that despite a decrease in total ^E amounts in rseA, there is an increase in the ratio of free ^E, as seen by changes in soluble versus membrane-bound fractions of rseA, rather than totally abolishing membrane association of RpoE.

View larger version (21K): [in this window] [in a new window]   Figure 4  E level and its intracellular location in rseA, rseB and rseC mutants. Cells were grown in LB medium at 37 °C with shaking until early stationary phase. (A) Total, (B) soluble and (C) inner membrane fractions were prepared as described in Experimental procedures. The samples were then subjected to 15% SDS–PAGE, followed by Western blotting with an antibody against E. As controls, (D) RseA- and (E) RseB-antibodies were used against the total fractions. (F) Represents Western blot from soluble fractions using antibody against RseB. (G) Shows the presence of ClpY in soluble (S) and inner membrane (IM) fractions from wild-type, rseA, rseB and rseC mutants used in the above panels. (H) Immunoblot showing detection of RpoE in the isogenic wild-type and waaA mutant in total crude cell extract, soluble fraction (S) and in the inner membrane fraction (IM), using equivalent amounts of proteins from each fraction. Arrows indicate the position of E, RseA, RseB and ClpY. The star symbol in (D) indicates cross-reacting RseA-specific cleavage product(s) in rseB mutant. Migrated prestained Molecular weight standards are shown.

View larger version (20K): [in this window] [in a new window]   Figure 5  Reduction in PpiD levels in rseA and suppression of cell lysis upon PpiD over-expression. (A) Equivalent amounts of inner membrane solubilized fractions from wild type (wt), rseA, rseB and rseC were applied to 12% SDS–PAGE and analyzed by Western blotting. Proteins blotted to nitrocellulose membrane were probed for the amounts of PpiD using anti-PpiD antibodies. (B) Logarithmic grown cultures of rseA transformed with empty vector pSE420 and isogenic rseA carrying cloned ppiD+ in pSE420 were adjusted to OD 0.05 and furthermore incubated with shaking at 37 °C. Cell lysis was measured as described in Fig. 1. The culture volume applied in case of rseA (empty vector) is 1 : 5, 1 : 10 and 1 : 20 as compared to that used for corresponding ppiD+ over-expressing in rseA, which was undiluted.

Control experiments were carried out using specific antibodies against RseA and RseB. Extracts from rseB showed accumulation of small anti-RseA cross-reacting species (marked with the star sign), which is consistent with RseB function in interaction with RseA, to protect it or alter its proteolysis (Fig. 4D). These data provide in vivo evidence of accumulation of RseA cleavage product(s), presumably due to its increased turnover in rseB mutants. Furthermore, RseB amounts seem to be increased only in rseA in both crude extracts and in soluble fractions, consistent with the negative regulation of whole rpoE operon by RseA (Fig. 4E,F). Moreover, no significant amounts of RseB in the IM fractions of the wild type, rseA or rseC could be detected to be physiologically relevant (data not shown).

Finally, our data demonstrate the accumulation of RseA cleavage product(s) in rseB mutants, explaining role for RseB acting as a modulator for RseA proteolysis in vivo.

Reduction in the amounts of PpiD in rseA and suppression of lysis in rseA mutants upon increased expression of PpiD, a peptidyl-prolyl isomerase with OMP folding function

To provide molecular basis of cell lysis phenotype, we examined if any OMP biogenesis factors in rseA mutants are limiting, which might explain this phenotype. The rationale being that rseA mutants or over-expression of RpoE in general leads to reduction in the levels of OMPs (Kabir et al. 2005). However, since the levels of HtrA, SurA, FkpA and Skp are increased in rseA mutants (Dartigalongue et al. 2001; Kabir et al. 2005), we looked for effect on other OMP biogenesis factors, which could be limiting and cause cell lysis. In this context role of the ppiD gene was examined, since its product recognizes early OMP folding intermediates and its over-expression suppresses OMP biogenesis defects of surA mutants restoring them to near wild-type levels (Dartigalongue & Raina 1998; Antonoaea et al. 2008). However, its levels have not been examined under the conditions of RpoE induction up to now. Thus, total cell extracts or fractionated samples from soluble and inner membrane fractions from wild-type or rseA/B/C mutants were analyzed. As shown, the levels of PpiD were severely reduced in rseA mutants if either total or in octyl-β-D-glucopyranoside solublized inner membrane (Fig. 5A). However, the levels of PpiD were unaffected by rseB or rseC mutations and were comparable to that in the wild type. Such a reduction in the levels of PpiD is up to now unprecedented in any genetic background and could partly account for OMP reduction and hence cell lysis in rseA mutants.

PpiD amounts were uniquely reduced only in rseA mutants, but not in rseB mutants. This reduction in the levels of PpiD in rseA mutants seems to mirror the cell lysis phenotype. Hence, we examined the influence of increased expression in trans of PpiD from a plasmid. The supernatant fractions from rseA mutant with empty vector and isogenic rseA construct, carrying the ppiD gene on the plasmid, were subjected to SDS–PAGE. Quite interestingly, the accumulation of protein was dramatically suppressed in the presence of PpiD plasmid (Fig. 5B). These results clearly suggest that upon multicopy plasmid-born expression of the ppiD gene, the cell lysis phenotype of rseA mutants is significantly suppressed and restored to nearly wild-type levels. PpiD multicopy suppression of cell lysis is reminiscent of suppression of OMP defects in a defined surA mutant by PpiD over-expression (Dartigalongue & Raina 1998). Thus, we can assume that cell lysis phenotype of rseA mutants arises mainly due to known reduction in the amounts of major OMPs, and PpiD over-expression causes suppression of this phenotype due to acceleration of OMP folding. These results indicate an integrated in built mechanism of cell lysis association with integrity of OM, which is presumably impaired in rseA mutants.

Reduction in VBNC population in rseA

The ^E-dependent cell lysis appears to occur specifically for VBNC cells that are defective in colony forming ability. Such cells become major in early stationary phase under usual growth conditions (Nitta et al. 2000; Kabir et al. 2004b). To attempt discrimination of the defective cells from the cells capable of forming colonies, morphological observation was carried out. The difference between the total count and the viable count shows the number of dead cells, and the difference between the viable count and CFU number shows the number of VBNC cells (Desnues et al. 2003; Abe et al. 2007). As depicted in Fig. 6, after the late log phase, cells loose the colony forming ability. The total cell count in the rseA was found to be decreased over days whereas that in wild type (WT), rseB or rseC did not show such significant reduction. The ratio between the total cell count and the viable cell count in rseA remained nearly constant over days. In contrast, the other strains increased this ratio. However, no major difference in the number of CFU was observed among all the strains. Therefore, rseA apparently reduced in the number of dead cells all along the incubation over days in contrast to other mutants and WT.

View larger version (15K): [in this window] [in a new window]   Figure 6  Determination of VBNC population in W3110 (WT), rseA, rseB and rseC strains. Cells were grown in LB medium at 37 °C with shaking. Cells were collected at times indicated and DAPI-stained all cells (open circles), CTC-stained viable cells (closed circles) and culturable cells (triangles) capable of forming a colony were determined as described in Experimental procedures. The experiments were carried out three times. Error bars represent standard deviation ± mean.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

The significant effect of the rseA mutation on cell lysis appears to be due to the increase in transcription of rpoE, and a significant decrease in the amounts of OMP biogenesis folding catalyst PpiD in rseA. Consistent with the lack of significant cell lysis in rseB mutants, only a minor decrease in the membrane association of ^E was observed. Lack of cell lysis phenotype of rseB is further supported by the absence of any major effect on PpiD levels and hence on OMP maturation. The ppiD gene was originally identified as a multicopy suppressor of surA mutants, which resulted in restoration of OMP biogenesis and maturation defects in such bacteria (Dartigalongue & Raina 1998). PpiD has recently been shown to be the first periplasmic factor that interacts with nascent secretory chains emerging from Sec machinery (Antonoaea et al. 2008). rseA have constitutively increased levels of RpoE-regulated genes, including negatively regulating OMP synthesis noncoding RNAs, accounting in part reduction in OMP levels upon RpoE induction (Valentin-Hansen et al. 2007). Furthermore, inactivation of the rseA gene concomitantly resulted in severe reduction of PpiD. Hence, the overall cell lysis phenotype in rseA due to membrane lysis would thus stem from coupled micA- and rybB-mediated repression, with further defective OMP folding due to reduced/limiting amounts of PpiD, explaining the cell lysis phenotype of rseA. The observed reduction in the levels of PpiD in rseA and repression of cell lysis in such mutants by PpiD over-expression provides substantial evidence that cell lysis arises due to OMP perturbations. PpiD-mediated suppression of cell lysis defect mimics similar suppression of OMP folding defects of surA mutants. However, this reduction in PpiD amounts is not unique to rseA, but is found in all conditions that induce RpoE activity significantly, particularly upon defects in OMP maturation, such as in deep-rough mutants and their underacylated derivatives (Klein et al. 2009). However, the magnitude of reduction in rseA is most severe. Our overall conclusion about OMP reduction due to reduced PpiD levels and induction of small non-coding RNA is further supported by a separate study, where we found that the knockout mutation of micA, encoding MicA for a regulator of ompA gene or rybB, encoding RybB for a regulator of ompC and ompW genes, also suppressed the ^E-dependent lysis and that of ompA, ompC or ompW caused cell lysis at a significant level (our unpublished results). Moreover, the lysis was enhanced when micA or rybB was over-expressed in the wild-type background. Therefore, we assumed that ^E, when at a high level, directs cell lysis by expressing micA and rybB, leading to reduction in OMP amounts, which is furthermore accentuated by accompanied reduction in PpiD assisted OMP folding and hence disintegration of the outer membrane.

The present study showed that despite increase in the rpoE transcription and RpoE activity, the overall amounts of RpoE do not increase in rseA mutants. To test the model that the primary cause and magnitude of induction of ^E-dependent promoters was due to increased ratio of soluble (accessible to RNA polymerase) versus IM form (sequestered), RpoE amounts were measured in waaA mutants. waaA mutants lack Kdo transferase and hence synthesize nonglycosylated free lipid IV_A minimal LPS, and show six- to eightfold increase in transcription from ^E-dependent promoters even under permissive growth conditions between 21 °C and 30 °C (Klein et al. 2009). Although waaA and rseA mutants are not exactly comparable, since the waaA gene is essential for bacterial growth above 30 °C, however, both the mutants have reduced OMP content and show high RpoE activity. Quite like rseA mutants, waaA mutants showed significantly decreased amounts of IM-associated form of RpoE, without any significant increase in total RpoE amount. Taken together, our results from rseA and waaA mutants provide a convincing mechanism of RpoE induction. It should be however noted that in waaA mutants soluble RpoE also showed a small increase, but that does not seem to correspond to six- to eightfold increase in the activity of ^E-dependent promoters. Thus, decreased inner membrane association, resulting in increase in ratio of soluble (free) versus IM form explains RpoE induction without increase of the total RpoE amounts. However, additional mechanisms implying IM association altering RpoE proteolysis or such an association causing abortive RNA polymerase association, which could be inhibitory cannot be ruled out and needs furthermore investigation.

Since the introduction of the rseB plasmid clone into the wild-type strain repressed both the accumulation of proteins in the medium in the wild-type strain as well rpoEP2 promoter activity, argues for a function for RseB as a modulator of ^E-dependent lysis in an RseA-dependent manner. However, this RseB repression required the functional presence of RseA, since neither of these processes could be repressed in rseA by RseB plasmid clone. Hence, when RseB was provided from the plasmid in the wild type, increase in RseB amounts could render RseA resistant to proteolytic action of DegS and RseP, leading to higher inhibition of ^E by its further sequestration than by the free RseA alone and consequent repression of cell lysis. Our in vivo results of accumulation of RseA proteolytic product(s) in rseB mutant thus provide a support for our conclusions and suggested models of RseB function based on in vitro results (Grigorova et al. 2004; Cezairliyan & Sauer 2006). Thus, it seems the function of RseB is primarily to modulate the RseA function and hence the effect on ^E.

Concerning RseC, significant amount of proteins accumulated in the rseC strain at 45 °C after 36-h incubation, which however may not be due to the ^E-dependent lysis, because increase in lysis seems to stop under the same condition in the rseA strain. Furthermore experiments are required to unravel the mechanism of the lysis in the rseC strain at the higher temperature as well as at intermediate temperature.

Although the VBNC populations in all the strains were nearly to the similar level, only rseA showed a significant reduction in dead cells compared to others. We assume that the VBNC population in rseA is directly subjected to cell lysis, rather than being the dead cell state. As a consequence, rseA showed a significant reduction in total cell count. However, in the WT, rseB or rseC mutant, both the dead cells and the VBNC cells increased gradually. At the higher temperature, further reduction of OD₆₀₀ and more amounts of proteins accumulated in the medium were observed in rseA, consistently with decrease in CFU, suggesting that damaged cells are accumulated as temperature increases.

Finally, it will be interesting to further understand the molecular basis of reduction of RpoE and PpiD amounts in rseA. It is particularly intriguing in case of RpoE, since the rpoE specific mRNA increases without corresponding increase in the total protein level.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Materials

Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo (Kyoto, Japan) and New England Biolabs. DNA sequencing kit (ABI PRISM^® BigDye^® Terminator v3.1 Cycle Sequencing Kit) was obtained from Applied Biosystems Japan. Oligonucleotide primers were synthesized by Proligo Japan K. K. (Tokyo, Japan). Other chemicals were all of analytical grade and obtained from commercial sources.

Bacterial strains, medium and culture conditions

The bacterial strains used in this study were derivatives of E. coli K-12. Their relevant genotypes and plasmids are presented in Table 1. Null mutations in the rseA, rseB, rseC genes with coding sequences replaced by kan cassette in the strain BW25113 were obtained from H. Mori (NIG Japan) and used as donors for bacteriophage P1-mediated transduction (Miller 1992) into W3110 genetic background. The removal of the kan cassette from each transduced strain was carried out as described below, resulting into strains YU692 (rseA), YU693 (rseB) and YU694 (rseC). Precultured cells were transferred into LB [1% (w/v) bactotryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl] medium, adjusted by dilution to an OD₆₀₀ of 0.1. Cells were further incubated for appropriate time at required temperatures under aerobic conditions by reciprocal shaking (100 times/min). When required, antibiotics were added as appropriate, at the following concentrations: ampicillin 50 µg/mL, tetracycline 8 µg/mL, kanamycin 25 µg/mL.

pCP20 is an Amp^R and Cml^R plasmid with a temperature-sensitive replication and thermal induction of FLP synthesis (Cherepanov & Wackernagel 1995). W3110 rseA::kan, W3110 rseB::kan or W3110 rseC::kan was transformed with pCP20, and the resultant ampicillin-resistant transformants were obtained at 30 °C, from which a few were colony-purified once nonselectively at 43 °C. Resultant derivatives were tested by PCR in addition to checking for the loss of all antibiotic resistances. The majority of deletion mutants lost simultaneously the FRT-flanked resistance gene and the FLP helper plasmid as described previously (Datsenko & Wanner 2000). A chromosomal deletion in the rpoE gene was constructed by substitution of its coding sequence by cat cassette from pKD3 (Datsenko & Wanner 2000).

DNA manipulation

Conventional recombinant DNA techniques were followed (Sambrook & Russell 2001). The DNA fragments bearing the coding region of rseA was amplified by PCR using the primers for rseA-5' (5'-GGGGGTACCGATAGCGGGATA-3') and rseA-3' (5'-GGGAAGCTTCCAAAGTTGCTTCATTACT-3'), rseB was amplified with the primers for rseB-5' (5'-GGGGGTACCCTG TACAGGTGCCAGGAATT-3') and rseB-3' (5'-GGGAAGCTT GATCATTGCGCTGCCCCGAA-3'), and rseC was amplified with the primers for rseC-5'(5'-GGGGGTACCCAAACGGCGA AACGCATTGC-3') and rseC-3' (5'-GGGCTGCAGATCACTG GCTCGCGTCTTCC-3') by PCR as described previously (Yamada et al. 1993), with KpnI or HindIII sites and W3110 genomic DNA as a template. The amplified DNA fragments bearing the rseA (650 bp), rseB (956 bp) or rseC (479 bp) genes were digested with KpnI and HindIII, and inserted between the KpnI and HindIII sites on pBAD24, generating pBADRSEA, pBADRSEB and pBADRSEC. The integrity of the cloned rseA, rseB or rseC genes was verified by nucleotide sequencing (Sanger et al. 1977). Each of the three genes was cloned under the control of araBAD promoter.

Monitoring of cell lysis

Cells were grown at various temperatures in the range of 30–45 °C as described above. The cell growth was monitored by measuring OD₆₀₀, and CFUs were determined by counting the colony number on LB plates after 24 h of incubation. Protein accumulation in medium was tested as described previously (Nitta et al. 2000). A portion of the culture was centrifuged at 3000 g for 10 min to separate the supernatant (medium fraction) and the pellet (remaining fraction). Proteins in the medium fraction were recovered by the addition of trichloroacetic acid at the final concentration of 5%, treated with ether and resolved in 20 mM Tris–HCl (pH 7.0). The pellet was resolved in 20 mM Tris–HCl (pH 7.0) and subjected to sonic oscillation. Proteins from both the fractions were resolved by 12% SDS–PAGE.

For the test for lysis, wild-type, rseA, rseB and rseC cells were grown at 42 °C as described above. A portion of the culture was centrifuged at 5000 g for 2 min to separate the supernatant (medium fraction) and the pellet (remaining fraction). The pellet fraction was suspended with Z-buffer and the β-galactosidase activity of both of the medium- and the remaining fractions were measured according to the procedure described by Miller (1992). The β-galactosidase activity in the medium fractions for each strain was divided by the sum of that of medium and remaining fractions.

Complementation studies

The rseA, rseB or rseC cloned in pBAD24 and the empty plasmid vector as a control were introduced into W3110 (wild type), W3110 rseA::Tn10Kan or W3110 rseA cells. The rseA plasmid clone or the empty plasmid vector was also introduced to W3110 rseB cells. The transformants were tested on LB plates containing ampicillin 50 µg/mL. Precultured cells were transferred into LB medium diluted at a turbidity corresponding to OD₆₀₀ of 0.1, and then the cells were further grown for appropriate times at 37 °C under aerobic conditions by reciprocal shaking (100 times/min). Protein accumulation in medium was measured as described above.

Transcriptional assay

For assay with the lacZ operon fusion, empty vector (as a control), rseA plasmid clone or rseB plasmid clone were transformed with the rpoE-lacZ fusion on the genome. Transformants were grown at 37 °C in LB media containing appropriate antibiotics. Exponentially grown culture was diluted with the same medium and adjusted to OD₆₀₀ of 0.02, and further incubated for the appropriate time. Samples were then taken from the culture, and β-galactosidase activity was measured according to the procedure described by Miller (1992).

RT-PCR analysis

Cells were grown in 30 mL of LB medium at different temperatures (37, 42 and 45 °C) for 10 h (early stationary phase), and total RNA was immediately extracted using the hot phenol method (Aiba et al. 1981). RT-PCR analysis was carried out using an mRNA selective RT-PCR Kit (Takara Shuzo) to examine the expression of rpoE (Tsunedomi et al. 2003). The primers used for rpoE expression were rpoE-5' (5'-TGGGGAGACTTTACCTC-3') and rpoE-3' (5'-TCGTCAACGCCTGATAA-3'). The RT reaction was carried out at 45 °C for 15 min using 0.1 µg of total RNA and each downstream primer, followed by PCR consisting of denaturing at 85 °C for 1 min, annealing at 45 °C for 1 min and extension at 72 °C for 2 min was carried out using the two primers for each gene. After the completion of 25, 30, 35 and 40 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 model GS-700 Imaging Densitometer (Bio-Rad) (Nitta et al. 2000). Linearity of the amplification was observed up to 30th or 35th cycle. In our conditions, the RNA-selective RT-PCR was able to specifically detect mRNA because no band was observed when reverse transcriptase was omitted.

Western blot analysis

Isogenic W3110, GK 2085 (rseA), GK 2086 (rseB) and GK 2087 (rseC) cells were grown at 37 °C for 10 h in 200 mL LB medium and harvested by centrifugation at 3000 g for 10 min. Cells were washed, resuspended in 10 mM Tris–HCl buffer (pH 8.0) and French pressed twice. The cell extracts were fractionated into the membrane and the soluble fractions by centrifugation at 17 000 g for 30 min (Yamada et al. 1993). To obtain inner membrane, proteins were extracted by incubation in 2% octyl-β-D-glucopyranoside. The solubilized inner membrane fraction was recovered by centrifugation at 17 000 g for 30 min. Crude extracts, membrane fractions and soluble fractions were applied to 15% SDS–PAGE. After the electrophoresis, proteins in the gel were blotted to nitrocellulose membrane. ^E, RseA, RseB, PpiD and ClpY proteins were detected with an antibody against ^E, RseA, RseB and ClpY as described previously (Yamada et al. 1993; Missiakas et al. 1996b; Dartigalongue & Raina 1998; Klein et al. 2003). For experiments with waaA mutant and its isogenic parent, cultures were grown at 21 °C in minimal M9 medium, washed and resuspended in LB medium at 30 °C and allowed to grow till OD of 0.4. Cells were harvested, fractionated as described above and analyzed by Western blot analysis for the presence of RpoE.

Determination of VBNC populations

The VBNC cell population was determined by the method described previously (Abe et al. 2007) with slight modification. Cells were grown as described above. The cell culturability (CFU) was measured by using LB agar plates. CFUs were enumerated with samples diluted at three different concentrations as described above. The staining procedure with DNA-binding fluorochrome, 4',6-Diaminido-2-phenylindole, dihydrochloride (DAPI, Dojindo Laboratories, Kumamoto, Japan) staining was used to count total bacterial without distinguishing between live and dead bacteria by using an epifluorescence microscope (Nikon, Eclipse, E-600) (Porter & Fieg 1980). Two per cent formalin-fixed samples were filtered through 0.2-µM-pore-size polycarbonate black filters (Toyo Roshi Kaisha, Ltd, Tokyo, Japan) and stained for 20 min with a 2.5 µg/mL DAPI solution. Viable cell counts were determined using the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Dojindo Laboratories) method (Rodriguez et al. 1992); that is, bacteria with a functioning electron transport chain reduce the CTC in CTC-formazan, forming a red fluorescent precipitate in the cell membrane. Samples were incubated with 3.0 mM CTC, in the presence of 0.025% yeast extract, overnight at room temperature, and then fixed with formalin (2% final concentration). Samples were stained with DAPI, as described above. Cells showing red precipitate under green excitation were counted. Development of VBNC was monitored using CFU enumeration and total and viable counts, as described above.

	Acknowledgements

 
We thank Dr. K. Matsushita for helpful discussion. This work was supported by a Grant-in-Aid for Basic Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.Y.), by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and support from FZB (to S.R. and G.K.).

	Footnotes

 
Communicated by: Hiroji Aiba

^* Correspondence: m-yamada{at}yamaguchi-u.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
Abe, A., Ohashi, E., Ren, H., Hayashi, T. & Endo, H. (2007) Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus. Microbiol. Res. 162, 130–138.

Aiba, H., Adhya, S. & de Crombrugghe, B. (1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J. Biol. Chem. 256, 11905–11910.[Abstract/Free Full Text]

Antonoaea, R., Fürst, M., Nishiyama, K. & Müller, M. (2008) The periplasmic chaperone PpiD interacts with secretory proteins exiting from the SecYEG translocon. Biochemistry 47, 5649–5656.[CrossRef][Medline]

Cezairliyan, B.O. & Sauer, R.T. (2006) Inhibition of regulated proteolysis by RseB. Proc. Natl. Acad. Sci. USA 104, 3771–3776.[CrossRef]

Cherepanov, P.P. & Wackernagel, W. (1995) Gene disruption in Escherichia coli: Tc^R and Km^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14.[CrossRef][Medline]

Collinet, B., Yuzawa, H., Chen, T., Herrera, C. & Missiakas, D. (2000) RseB binding to the periplasmic domain of RseA modulates the RseA: ^E interaction in the cytoplasm and the availability of ^E•RNA polymerase. J. Biol. Chem. 275, 33898–33904.[Abstract/Free Full Text]

Costanzo, A. & Ades, S.E. (2006) Growth phase-dependent regulation of the extracytoplasmic stress factor, ^E, by guanosine 3',5'-bispyrophosphate (ppGpp). J. Bacteriol. 188, 4627–4634.[Abstract/Free Full Text]

Cuny, C., Dukan, L., Fraysse, L., Ballesteros, M. & Dukan, S. (2005) Investigations of the first events leading to the loss of culturability during Escherichia coli starvation: Future nonculturable bacteria form a subpopulation. J. Bacteriol. 187, 2244–2248.[Abstract/Free Full Text]

Dartigalongue, C., Missiakas, D. & Raina, S. (2001) Characterization of the Escherichia coli ^E regulon. J. Biol. Chem. 276, 20866–20875.[Abstract/Free Full Text]

Dartigalongue, C. & Raina, S. (1998) A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli. EMBO J. 17, 3968–3980.[CrossRef][Medline]

Datsenko, K.A. & Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645.[Abstract/Free Full Text]

De Las Peñas A., Connolly, L. & Gross, C.A. (1997a) ^E is an essential sigma factor in Escherichia coli. J. Bacteriol. 179, 6862–6864.[Abstract/Free Full Text]

De Las Peñas, A., Connolly, L. & Gross, C.A. (1997b) the ^E-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of ^E. Mol. Microbiol. 24, 373–385.[CrossRef][Medline]

Desnues, B., Cuny, C., Grégori, G., Dukan, S., Aguilaniu, H. & Nyström, T. (2003) Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells. EMBO Rep. 4, 400–404.[CrossRef][Medline]

Erickson, J.W. & Gross, C.A. (1989) Identification of the ^E subunit of Escherichia coli RNA polymerase: a second alternative s factor involved in high-temperature gene expression. Genes Dev. 3, 1462–1471.[Abstract/Free Full Text]

Grigorova, I.L., Chaba, R., Zhong, H.J., Alba, B.M., Rhodius, V., Herman, C. & Gross, C.A. (2004) Fine-tuning of the Escherichia coli ^E envelope stress response relies on multiple stress response relies on multiple mechanisms to inhibit signal-independent preoteolysis of the transmembrane anti-sigma factor, RseA. Genes Dev. 18, 2686–2697.[Abstract/Free Full Text]

Hazan, R., Sat, B. & Engelberg-Kulka, H. (2004) Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. J. Bacteriol. 186, 3663–3669.[Abstract/Free Full Text]

Hiratsu, K., Amemura, M., Nashimoto, H., Shinagawa, H. & Makino, K. (1995) The rpoE gene of Escherichia coli, which encodes ^E, is essential for bacterial growth at high temperature. J. Bacteriol. 177, 2918–2922.[Abstract/Free Full Text]

Kabir, M.S., Sagara, T., Oshima, T., Kawagoe, Y., Mori, H., Tsunedomi, R. & Yamada, M. (2004a) Effects of mutations in the rpoS gene on cell viability and global gene expression under nitrogen starvation in Escherichia coli. Microbiology 150, 2543–2553.[Abstract/Free Full Text]

Kabir, M.S. & Yamada, M. (2005) ^E-dependent programmed cell death in Escherichia coli. In: Yamada M, ed. Survival and Death in Bacteria, Research Signpost, Trivandrum, India, pp. 55–67.

Kabir, M.S., Yamashita, D., Koyama, S., Oshima, T., Kurokawa, K., Maeda, M., Tsunedomi, R., Murata, M., Wada, C., Mori, H. & Yamada, M. (2005) Cell lysis directed by ^E in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli. Microbiology 151, 2721–2735.[Abstract/Free Full Text]

Kabir, M.S., Yamashita, D., Noor, R. & Yamada, M. (2004b) Effect of ^S on ^E-directed cell lysis in Escherichia coli early stationary phase. J. Mol. Microbiol. Biotechnol. 8, 189–194.[CrossRef][Medline]

Klein, G., Dartigalongue, C. & Raina, S. (2003) Phosphorylation-mediated regulation of heat shock response in Escherichia coli. Mol. Microbiol. 48, 269–285.[CrossRef][Medline]

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.[Abstract/Free Full Text]

Koo, M.S., Lee, J.H., Rah, S.Y., Yeo, W.S., Lee, J.W., Lee, K.L, Koh, Y.S., Kang, S.O. & Roe, J.H. (2003) A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J. 22, 2614–2622.[CrossRef][Medline]

Miller, J.H. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Missiakas, D., Betton, J.M. & Raina, S. (1996a) New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol. Microbiol. 21, 871–884.[CrossRef][Medline]

Missiakas, D., Mayer, M.P., Lemaire, M., Georgopoulos, C. & Raina, S. (1997) Modulation of the Escherichia coli ^E (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins. Mol. Microbiol. 24, 355–371.[CrossRef][Medline]

Missiakas, D. & Raina, S. (1997) Protein misfolding in the cell envelope of Escherichia coli: new signaling pathways. Trends Biochem. Sci. 22, 59–63.[CrossRef][Medline]

Missiakas, D., Schwager, F., Betton, J.M., Georgopoulos, C. & Raina, S. (1996b) Identification and characterization of HslV HslU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J. 15, 6899–6909.[Medline]

Nitta, T., Nagamitsu, H., Murata, M., Izu, H. & Yamada, M. (2000) Function of the ^E regulon in dead-cell lysis in stationary-phase Escherichia coli. J. Bacteriol. 182, 5231–5237.[Abstract/Free Full Text]

Nyström, T. (2003) Conditional senescence in bacteria: death of the immortals. Mol. Microbiol. 48, 17–23.[CrossRef][Medline]

Porter, K.G. & Fieg, Y.S. (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25, 943–948.

Raina, S., Missiakas, D. & Georgopoulos, C. (1995) The rpoE gene encoding the ^E (²⁴) heat shock sigma factor of Escherichia coli. EMBO J. 14, 1043–1055.[Medline]

Rodriguez, G.G., Phipps, D., Ishiguro, K. & Ridgway, H.F. (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58, 1801–1808.[Abstract/Free Full Text]

Rouvière, P.E., De Las Peñas, A., Mecsas, J., Lu, C.Z, Rudd, K.E. & Gross, C.A. (1995) rpoE, the gene encoding the second heat-shock sigma factor, ^E, in Escherichia coli. EMBO J. 14, 1032–1042.[Medline]

Sambrook, J. & Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.[Abstract/Free Full Text]

Tsunedomi, R., Izu, H., Kawai, T., Matsushita, K., Ferenci, T. & Yamada, M. (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.[Abstract/Free Full Text]

Valentin-Hansen, P., Johansen, J. & Rasmussen, A.A. (2007) Small RNAs controlling outer membrane porins. Curr. Opin. Microbiol. 10, 152–155.[CrossRef][Medline]

Walker, G.C. (1996) The SOS response of Escherichia coli. In: Neidhardt FC, Curtiss RI, Ingraham JL, Lin CCL, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE, eds. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, DC, pp. 1400–1416.

Wang, Q.P. & Kaguni, J.M. (1989) A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli. J. Bacteriol. 171, 4248–4253.[Abstract/Free Full Text]

Yamada, M., Sumi, K., Matsushita, K., Adachi, O. & Yamada, Y. (1993) Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site. J. Biol. Chem. 268, 12812–12817.[Abstract/Free Full Text]

Received: 8 March 2009
Accepted: 27 April 2009

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Noor, R. Articles by Yamada, M. Search for Related Content PubMed Articles by Noor, R. Articles by Yamada, M.