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E is involved in responses to cell surface stresses and its activity is controlled by the anti- factor CseE
1 Graduate School of Biotechnology, Korea University, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea
2 Institute of Biotechnology 1, Heinrich Heine University, Research Center Jülich, D-52425 Jülich, Germany
3 Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Chungnam 339-700, Republic of Korea
4 Department of Oriental Medicine, Semyung University, Checheon, Chungbuk 390-230, Republic of Korea
Correspondence
Heung-Shick Lee
hlee{at}korea.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E, an alternative factor of Corynebacterium glutamicum, is involved in cell surface stresses. Cells in which the sigE gene was deleted evidenced increased sensitivity to magnesium deficiency, as well as to SDS, lysozymes, EDTA and heat. We utilized physiological analyses to show that the downstream gene, designated cseE, encodes an anti- factor. The retarded growth of the cseE mutant cells under ordinary growth conditions could be recovered by an additional deletion of sigE encoding E. Under stress conditions, the phenotype of the cseE-overexpressing cells mimicked that of the sigE mutant. The sigE and cseE genes were transcribed into a single transcript, and gene transcription was stimulated by heat. The SigE and CseE proteins interacted physically in vitro, in the form of glutathione S-transferase (GST) and maltose binding protein (MBP) fusion proteins, respectively. 2D-PAGE analysis of the wild-type and mutant crude extracts showed that the sigE mutant failed to synthesize a 34 kDa polypeptide that was normally induced in wild-type cells grown under heat (or SDS)-stressed conditions. The protein turned out to be expressed from ORF NCgl1070 and showed similarity to methyltransferases which may confer resistance to antibiotics. Accordingly, the sigE mutant evidenced extreme sensitivity to antibiotics, including nalidixic acid, penicillin and vancomycin. Finally, we present a discussion of the possible role of the sigE and cseE genes in the acclimation of C. glutamicum to cell surface stress conditions.
Present address: Institute of Molecular Microbiology and Biotechnology, Westphalian Wilhelms University Muenster, Corrensstr. 3, 48149 Muenster, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kalinowski et al., 2003) has made it possible to characterize the genetic background of the organism in a new dimension.
The correct regulation of gene expression in a given organism is crucial for adaptation and survival under a variety of growth conditions. This is achieved, in part, by the reversible association of different factors with bacterial RNA polymerase to modulate the transcription of the target genes (Browning & Busby, 2004; Gruber & Gross, 2003). Bacterial genomes may encode a principal factor which is dedicated to the transcription of housekeeping genes, and may also harbour a variety of alternative factors which participate in the coordination of gene expression during a variety of stress responses as well as morphological development. The extracytoplasmic function (ECF) factors constitute a class of alternative factors (Helmann, 2002), and the genes that encode these ECF factors have been identified in a variety of organisms, although the roles and mechanisms inherent to regulation are only now coming to light. At least 10 putative genes that encode ECF factors have been identified within the genome of Mycobacterium tuberculosis (Rodrigue et al., 2006), and 51 genes in Streptomyces coelicolor (Hahn et al., 2003; Lee et al., 2005).
In the C. glutamicum genome, a total of seven putative factor genes have been detected (Brinkrolf et al., 2007; Engels et al., 2004; Kalinowski et al., 2003). The sigA gene encodes the essential primary factor of C. glutamicum and is also responsible for the promoter recognition of housekeeping genes (Halgasova et al., 2001; Oguiza et al., 1996). The alternative factor SigB replaces SigA during transition from exponential growth to stationary phase, and modulates the expression of a number of genes (Larisch et al., 2007). In addition to sigA and sigB, the annotation of the genome sequence of C. glutamicum ATCC 13032 has revealed five ECF factor genes, sigC, sigD, sigE, sigH and sigM (Engels et al., 2004; Kim et al., 2005; Nakunst et al., 2007), and several anti- factor genes (Pátek, 2005). Although factors have been shown to regulate gene transcription profiles in a simple mechanism involving the modulation of factor levels, the activity of certain factors can be regulated further by antagonistic proteins. Anti- factors interact directly with specific factors. The sequestration of factors blocks their association to RNA polymerases, thus preventing the transcription of target genes until an appropriate environmental stimulus is applied to the bacterium.
Among the ECF factor genes, only sigH and sigM have been studied to some extent thus far. The factors are involved in responses to thiol-oxidative stress and constitute a hierarchical regulatory network (Kim et al., 2005; Nakunst et al., 2007). In this study, we examined the sigE gene in detail and generated evidence to suggest that the sigE gene of C. glutamicum is involved in surface stress responses. In addition, we present evidence that SigE interacts with its cognate anti- factor, which is referred to as CseE. This is, to the best of our knowledge, the first report in C. glutamicum regarding the role of sigE as well as the presence and interaction of an anti- factor with its cognate factor.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli DH5
F' was utilized for the construction and propagation of plasmids. E. coli BL21 DE3 was employed for the expression of sigE and cseE cloned into pMAL-c2 and pGEX-4T-3, respectively. Unless otherwise stated, E. coli and C. glutamicum cells were cultured at 37 °C in Luria broth (Sambrook & Russell, 2001) and at 30 °C in MB medium (Follettie et al., 1993), respectively. Minimal media for C. glutamicum were based on MCGC (von der Osten et al., 1989). Carbon source glucose was added to the minimal medium at a concentration of 1 % (w/v). Antibiotics were added at the following concentrations: 50 µg ampicillin ml–1; 20 µg chloramphenicol ml–1; and 20 µg kanamycin ml–1. X-Gal was added to the media at a concentration of 40 µg ml–1. For protein expression, IPTG was added to a final concentration of 1 mM.
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). Plasmids were introduced into C. glutamicum cells via electroporation (Follettie et al., 1993), and mini-plasmid preparation for C. glutamicum cells was conducted as described by Yoshihama et al. (1985). PCR was carried out as previously described (Kim et al., 2005).
Plasmid construction and gene inactivation.
The pSL419 and pSL420 plasmids were constructed via the crossover PCR method (Hwang et al., 2002; Link et al., 1997). The primers utilized herein are listed in Table 1
. The primary PCR products, AB and CD, were amplified with 600 nM of outer primers and 60 nM of inner primers. These products were then directly utilized as templates for secondary PCR with 600 nM of outer primers. The secondary PCR products were ligated into the BamHI site of pK19mobsacB. The fragment was then introduced into SalI- and SmaI-digested pK19mobsacB, and the resultant plasmid was introduced into C. glutamicum AS019E12 in order to delete chromosomal sigE and/or cseE. Subsequent steps were conducted as described previously (Hwang et al., 2002; Schäfer et al., 1994), and the chromosomal deletion of sigE and/or cseE in C. glutamicum AS019E12 was confirmed via PCR (data not shown). The pSL418 plasmid was constructed via the amplification of the cseE gene using the cseE-oe1 and cseE-oe2 primers, and by subsequently ligating the amplified DNA into the PstI site of pSL360. The pSL421 plasmid was constructed via the amplification of the sigE gene using the GSTSigE1 and GSTsigE2 primers, and by subsequently ligating the amplified DNA with the BamHI/PstI-digested pGEX-4T-3 vector. The pSL422 plasmid was constructed by the same scheme with the exception of the primers, which were MALCseE1 and MALCseE2.
RNA analysis.
RNA was purified with TRIzol reagent (Invitrogen), followed by the RNeasy Mini kit (Qiagen) and RNase-Free DNase set (Qiagen). RT-PCR analysis for the sigE and cseE genes was conducted using the oligonucleotides listed in Table 1. First-strand cDNA was synthesized with the SuperScript III kit (Invitrogen) in accordance with the manufacturer's instructions. A 1 µl aliquot of a first-strand cDNA reaction mixture was employed as a PCR template. Each of the 20 cycles was conducted for 20 s at 95 °C, 30 s at 58 °C, and 45 s at 72 °C. The reaction products were then electrophoretically separated on agarose gel and visualized upon ethidium bromide staining.
Protein purification and analysis.
The glutathione S-transferase (GST)–SigE and maltose binding protein (MBP)–CseE fusion proteins were expressed and purified with the pGEX-4T-3 and pMAL-c2 vectors, respectively, in accordance with the manufacturer's instructions. The cell extracts were prepared as described by Follettie et al. (1993). The purified proteins were divided into aliquots and maintained at –70 °C. Proteins were measured via the Bradford method, with BSA as the standard (Bradford, 1976). SDS-PAGE and Western blot analysis were conducted as described elsewhere (Laemmli, 1970). 2D-PAGE was conducted as follows. First, IEF was conducted on an Ettan IPGphor II system (GE Healthcare), in accordance with the manufacturer's instructions. Second-dimension analyses were conducted on Protean II vi cells (Bio-Rad). The proteins were visualized via silver staining using a kit from Bio-Rad. Peptide analysis of the protein spots was performed by a commercial service (In2gen) via electrospray ionization MS (ESI-MS).
Interaction of GST–SigE and MBP–CseE.
Partially purified proteins (30 µg each) were incubated for 1 h at 25 °C in binding buffer [50 mM Tris/HCl, 0.1 M NaCl, 1 mM DTT, 10 % (v/v) glycerol, 1 % (v/v) Triton X-100, pH 7.5] and loaded onto a Glutathione Sepharose 4B column (GE Healthcare). The column was washed with 10 volumes washing buffer [50 mM HEPES, 0.15 M NaCl, 0.1 % (v/v) Tween 20, pH 7.5]. Finally, the bound proteins were eluted with elution buffer (0.1 M Tris/HCl, 0.1 M NaCl, 20 mM glutathione, pH 8.0). Aliquots (20 µl) were subjected to 12 % (v/v) SDS-PAGE. The protein bands were visualized by Coomassie Blue staining.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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sigE, cseE, and sigE/cseE mutants have reported that ORF NCgl1075 (cg1271) of C. glutamicum encodes an ECF factor, E. The encoded SigE protein is composed of 213 aa, and appears to be a member of the Group 4 proteins, which harbour the conserved regions 2 and 4 (Gruber & Gross, 2003). factors in Group 4, which are also often referred to as ECF factors, are often involved in response to stress conditions, including oxidative stress and surface stress (Alba & Gross, 2004). The amino acid sequence identities of the C. glutamicum SigE with those of the known SigE proteins from the closely related organisms M. tuberculosis and S. coelicolor were 63 and 42 %, respectively. Downstream of the sigE gene, the ORF NCgl1076 (cg1272), encoding a 164 aa polypeptide, was located. The ORF harboured an HX3CX2C motif that is found in many anti- factors (data not shown). In a variety of organisms, the sigE locus is conserved and the anti- factor gene is located immediately downstream of its cognate factor gene. Therefore, ORF NCgl1076 was designated cseE (control of sigE). Downstream of the cseE gene, the tatB gene encoding the twin arginine translocase protein was located.
In order to determine the possible functions of and relationships between SigE and CseE, the corresponding genes of C. glutamicum were deleted via gene disruption, as described in Methods. The constructed mutant strains were grown on minimal media, and the phenotypic changes of C. glutamicum mutants were assessed. As shown in Fig. 1, the growth of the sigE mutant was comparable to that of the parental strain, evidencing a doubling time of 
3 h. This suggests that the gene plays a non-essential role under ordinary growth conditions. However, the cseE mutant strain evidenced retarded growth, manifesting a doubling time of approximately 6 h. To our surprise, when we additionally deleted the sigE gene of the cseE mutant strain, the growth rate of the constructed sigE/cseE double mutant strain recovered to almost the parental level (Fig. 1). This indicates an antagonistic relationship between cseE and sigE, and suggests a possible interaction of the sigE and cseE gene products in the cell as a factor and its cognate anti- factor (see below).
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sigE, cseE and sigE/cseE mutants factors are known to be involved in responses to environmental stress conditions, we challenged the parental strain and the constructed mutants with a variety of environmental stresses and compared the growth properties. As the sigE genes of other organisms are known to be involved in cell surface stress responses, we initially administered heat challenge to the constructed mutants. In the case of the parental AS019E12 strain, 77 and 72 % cells survived after 3 and 5 h of heat treatment at 42 °C (Fig. 2), respectively. However, in the case of the sigE mutant, only 53 and 42 % of the cells survived after the same treatment. Mutants harbouring the deleted cseE gene evidenced growth comparable to that of the parental strain, with 76 % survival after 3 h. In the case of the sigE/cseE double mutant strain, the survival rate was 52 %. Surprisingly, when the cseE gene was overexpressed via the introduction of pSL418 into the wild-type strain, the sensitivity of the recombinant cells to heat increased to levels comparable to those observed with the sigE mutant strain. Comparable results were observed when the cells were challenged with anionic detergent, such as 0.01 % SDS (Table 2). No increased sensitivities of the mutants were observed in conjunction with other types of stress, including diamide oxidation stress, NaCl or KCl for salt stress, and pH stress (data not shown). These data show that the sigE gene is involved in responses to detergents and heat, and that the cseE gene likely antagonizes the activity of sigE.
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sigE and sigE/cseE mutants evidenced only 20 % of the growth seen in the parental strain (Table 2). The addition of 4 mM EDTA to the ordinary minimal medium also resulted in a retardation of the growth of the sigE mutant strain, but not the parental strain (Table 2). We also evaluated the effects of lysozymes on the growth of the mutant cells. As shown in Table 2, the growth of the sigE and sigE/cseE mutants was substantially affected by <0.5 µg lysozyme ml–1, whereas the growth of the parental strain was marginally affected. For an unknown reason, the cseE mutant strain survived to a greater degree than the parental strain under these conditions. Similar survival patterns were observed when the cells were challenged with the cell wall lytic enzyme mutanolysin (data not shown). Consequently, these data show not only the involvement of the sigE gene in the maintenance of cell envelope integrity, but also the role of cseE in antagonizing the action of the sigE gene.
Expression of the sigE and cseE genes
Knowing the interaction of the sigE and cseE genes, we assessed the possible organization of the genes in an operon. As expected, a single transcript of 1325 bp was detected in an RT-PCR experiment (Fig. 3), thereby indicating that the two genes are transcribed in a single transcription unit. Within the normal cellular propagation period, the sigE–cseE transcripts were expressed only marginally in the wild-type strain. However, upon heat treatment, the quantity of the sigE–cseE transcripts was increased significantly.
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factor and its cognate anti- factor, respectively. As shown in Fig. 1, under ordinary growth conditions, the deletion of the cseE gene induced a retardation of cellular growth, and the phenotype was recovered via the introduction of an additional sigE deletion. Furthermore, under stress conditions, cells that overexpressed the cseE gene mimicked the phenotype of the sigE mutant strain, thereby suggesting the possible titration of SigE by CseE. In order to analyse directly a possible interaction of the two proteins, we partially purified the proteins as MBP (42 kDa) or GST (27 kDa) fusion proteins, and then analysed the protein–protein interactions in vitro. As is shown in Fig. 4 (lanes 3 and 4), the two proteins of GST–SigE (27 kDa+24 kDa=51 kDa) and MBP–CseE (42 kDa+17 kDa=59 kDa) formed protein–protein complexes, as is evidenced by the coelution of the two proteins. The presence or absence of DTT or diamide had no effect on the interaction (data not shown). These data indicate that the antagonistic activity of CseE against SigE (Fig. 2, Table 2) is exerted via protein–protein interaction. It also indicates that the sigE-mimicking phenotype of the cseE-overexpressing cells was induced by the titration of SigE by CseE proteins.
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factor and its cognate anti- factor, we attempted to identify genes under the control of SigE via analyses of the total cell proteins using 2D gel electrophoresis. We searched for proteins present in the wild-type cells but not in the sigE mutant strain, after treating cells with heat or SDS. After careful scrutiny of the silver-stained gels, we identified a protein spot consistent with our guidelines. As shown in Fig. 5(a), the protein with a pI of 5.4 and Mr of 34 000 was detected in the heat- or SDS-treated wild-type cells but not in the sigE mutant, regardless of heat or SDS treatment. In accordance with our expectations, the protein was not detected in a cseE-overexpressing strain (data not shown). The amino acid sequences of the internal peptides of the protein (QGYVTLAGGAGLR, VGAVIADAWAR, DLVEFEMLLDQK), which were obtained via ESI-MS, matched that of the ORF NCgl1070-encoded protein of C. glutamicum ATCC 13032. The ORF (also referred to as cg1266) encoded a protein with an estimated molecular mass of 30 805 Da and a pI of 5.27, which are quite consistent with our 2D-PAGE data. In addition, the encoded protein showed similarity to a putative methyltransferase of Corynebacterium jeikeium (YP 251177), a putative mycinamicin-resistant protein of Corynebacterium efficiens (NP 737782) and a putative macrolide-resistance protein of Corynebacterium diphtheriae (NP 939350). Interestingly, the ORF was located in the close vicinity of the sigE and cseE loci (data not shown). We also analysed the expression profiles of NCgl1070 by monitoring the mRNA level. Consistent with the 2D-PAGE data, the mRNA of the ORF NCgl1070 was only detected in the heat-stressed wild-type cells (Fig. 5b). We also identified several other protein spots which appeared to be under the control of sigE. However, these proteins were not inducible by heat or SDS; therefore, the proteins were excluded from the present study (data not shown).
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sigE mutant to antibiotics; Dubnau et al., 2000) or by methylating the ribosomal content (Wachino et al., 2006). Knowing that the ORF NCgl1070, which may encode methyltransferases, is under the control of sigE, we assessed possible sensitivity of the sigE mutants to various antibiotics. As shown in Fig. 6, in the presence of antibiotics such as nalidixic acid, penicillin and vancomycin, the sigE mutants evidenced poor growth compared to the wild-type strain, whereas the growth of the cseE mutant strain was affected only marginally, considering the intrinsic slow growth of the cseE mutant strain. In addition, compared to the wild-type strain, the sigE mutant showed increased sensitivity to antibiotics such as tetracycline, ampicillin, chloramphenicol and ethambutol (data not shown). These data may indicate that the increased sensitivity of the sigE mutant cells to such antibiotics with different roles is attributable to the increased permeability of the cell envelope.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E of C. glutamicum evidences functional similarity to that of M. tuberculosis, the sigE of which is probably induced after exposure to heat shock, the detergent SDS, and vancomycin (Rodrigue et al., 2006). In this bacterium, the disruption of the sigE gene results in an increased sensitivity of the cells to those agents. In S. coelicolor, SigE is also required for normal cell integrity, although an anti- factor is not involved in controlling the activity (Hutchings et al., 2006; Paget et al., 2001). The sensitivity of the C. glutamicum sigE mutant cells to antibiotics and detergents may be attributable to the absence of methyltransferases, the expression of which is controlled by sigE, as shown in this study. In accordance with our assumption, in the related species M. tuberculosis, the S-adenosylmethionine methyltransferase (SAM-MT) protein has been demonstrated to catalyse key chemical modifications in defined positions of mycolic acids, thereby modifying the permeability of the cell envelope and conferring increased resistance to environmental agents, including antibiotics (Boissier et al., 2006; Dubnau et al., 2000). Ikeda and Nakagawa (2003) and Kalinowski et al. (2003) annotated NCgl1070 (cg1266) as encoding SAM-MT and rRNA guanine-N1-methyltransferase, respectively. Since the sigE mutant strain showed sensitivity to antibiotics that not only affect cell wall integrity but also inhibit cellular translation processes, it appears more likely that cg1266 encodes a methyltransferase, such as SAM-MT, that affects cellular permeability. The location of cg1266, in close proximity to the sigE/cseE locus, also supports this notion. In addition, other genes clustered within this region appear to be involved in cell envelope biosynthesis.
As shown in this study, the E of C. glutamicum appears to play roles distinctly different from those of H. The H and M of C. glutamicum are involved in oxidative and heat-stress responses (Engels et al., 2004; Kim et al., 2005; Nakunst et al., 2007), whereas E is involved in cell envelope stresses. In this regard, the sensitivity of the sigE mutants to heat may be attributed to increased membrane fluidity, thus allowing the entry of harmful extracellular substances. In addition, although SDS generally causes damage to membrane structures, it has also been suggested that SDS induces lipid peroxidation and the induction of the oxidative stress response, thus inducing genes associated with thioredoxin, glutathione and glutaredoxin (Nickerson & Aspedon, 1992; Singer & Tjeerdema, 1993; Sirisattha et al., 2004). However, this does not appear to be the case for the sigE gene of C. glutamicum, as the C. glutamicum sigE mutant did not evidence sensitivity to thiol-oxidative agents, including diamide. In C. glutamicum, the sigH and sigM genes participate in responses to oxidative stress (Kim et al., 2005; Nakunst et al., 2007).
The SigE protein of C. glutamicum is likely antagonized by its cognate anti- factor CseE. In this study, we have presented evidence that the cseE gene product of C. glutamicum functions as an anti- factor for E. This was demonstrated by the following: (1) the cseE mutant phenotype could be recovered by the introduction of an additional deletion of sigE; (2) the cseE-overexpressing cells evidenced a sigE mutant phenotype under stress conditions; (3) the SigE and CseE proteins interacted physically in vitro; and (4) there was physical linkage of the genes, which were expressed as a single transcription unit. The basis of the anti- factor inhibition of the function of the factor is the reversible protein–protein interaction of the anti- factor with its cognate factor (Duncan & Losick, 1993; Hughes & Mathee, 1998). CseE of C. glutamicum, a protein of 164 aa, harbours four conserved cysteine residues, all of which might be essential for CseE to inhibit the activity of SigE. As suggested for S. coelicolor (Bae et al., 2004; Kang et al., 1999; Paget et al., 2001) and M. tuberculosis (Rodrigue et al., 2006; Song et al., 2003), the cysteine may perform important functions in controlling the activity of CseE. In S. coelicolor, the cysteine residues of the RsrA protein, an anti- factor for SigR, are coordinated under reducing conditions by zinc. Upon oxidation, disulfide bonds are formed among the cysteine residues to induce conformational changes in the protein, thus releasing the factor. In this bacterium, the HCC motif contributes to zinc coordination. It will be interesting to determine whether or not the CseE protein of C. glutamicum, which also harbours the HCC motif, also participates in the coordination of zinc. Furthermore, the signal and the potential effectors involved in the release of SigE by CseE are currently unknown, and remain to be elucidated.
C. glutamicum evidences intrinsic resistance to certain antibiotics, including nalidixic acid. The presence of long-chain -alkyl, β-hydroxy fatty acids, the so-called mycolic acids, in the cell wall contributes significantly to the permeability of the cell wall (Daffé, 2005; Liu et al., 1996). The sigE mutant strain evidenced elevated sensitivity to a variety of antibiotics, including nalidixic acid, penicillin and vancomycin. Penicillin inhibits the transpeptidation reaction by binding to the transpeptidase enzymes involved in peptidoglycan biosynthesis, and vancomycin inhibits transpeptidation by binding to the terminal D-Ala-D-Ala residues of uncross-linked peptides present in the peptidoglycan (Hubbard & Walsh, 2003). In addition, the sigE mutant cells were more sensitive to lysozymes, which attack the peptidoglycan layer. These data may indicate the disruption of the peptidoglycan sacculus in the sigE mutant strain, thus indicating a role for the sigE gene in both peptidoglycan biosynthesis and osmoprotection. However, we were able to dismiss the possibility of an osmoprotective role for sigE, as the growth properties of the sigE mutant cells were indistinguishable from those of the parental strain when exposed to high-salt conditions (data not shown). Considering that the mutant cells were also sensitive to nalidixic acid, which affects DNA synthesis via the inhibition of DNA gyrase, a topoisomerase that induces the negative supercoiling of DNA (Cozzarelli, 1980; Gellert, 1981), we can conclude that the increased sensitivity of the sigE mutant cells to such antibiotics with different roles appears to be attributable to the increased permeability of the cell envelope, thus defining the role of sigE-controlled genes in the synthesis of cell surface structures to protect cells against cell surface stresses.
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ACKNOWLEDGEMENTS |
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Edited by: J.-H. Roe
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Received 24 August 2007;
revised 20 November 2007;
accepted 21 November 2007.
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