5FNC1†Present address: Institute of Molecular Microbiology and Biotechnology, Westphalian Wilhelms University Muenster, Corrensstr. 3, 48149 Muenster, Germany.
In this study, we demonstrate that σE, an alternative σ 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-σ factor. The retarded growth of the cseE mutant cells under ordinary growth conditions could be recovered by an additional deletion of sigE encoding σE. 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.
BradfordM. M.1976; A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
DafféM.2005; The cell envelope of corynebacteria. In Handbook of Corynebacterium glutamicum pp 121–148 Edited by
EggelingL.,
BottM.
Boca Raton, FL: CRC Press;
DuncanL.,
LosickR.1993; SpoIIAB is an anti- σ factor that binds to and inhibits transcription by regulatory protein σ F from Bacillus subtilis . Proc Natl Acad Sci U S A 90:2325–2329
EngelsS.,
SchweitzerJ. E.,
LudwigC.,
BottM.,
SchafferS.2004; clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σ H
. Mol Microbiol 52:285–302
HalgasovaN.,
BukovskaG.,
TimkoJ.,
KormanecJ.2001; Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB , from Brevibacterium flavum . Curr Microbiol 43:249–254
HutchingsM. I.,
HongH. J.,
LeibovitzE.,
SutcliffeI. C.,
ButtnerM. J.2006; The σ E cell envelope stress response of Streptomyces coelicolor is influenced by a novel lipoprotein, CseA. J Bacteriol 188:7222–7229
IkedaM.,
NakagawaS.2003; The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109
KalinowskiJ.,
BatheB.,
BartelsD.,
BischoffN.,
BottM.,
BurkovskiA.,
DuschN.,
EggelingL.,
EikmannsB. J.other authors2003; The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25
LarischC.,
NakunstD.,
HüserA. T.,
TauchA.,
KalinowskiJ.2007; The alternative sigma factor SigB of Corynebacterium glutamicum modulates global gene expression during transition from exponential growth to stationary phase. BMC Genomics 8:4
LeeE. J.,
KaroonuthaisiriN.,
KimH. S.,
ParkJ. H.,
ChaC. J.,
KaoC. M.,
RoeJ. H.2005; A master regulator σ B governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor . Mol Microbiol 57:1252–1264
LinkA. J.,
PhillipsD.,
ChurchG. M.1997; Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli : application to open reading frame characterization. J Bacteriol 179:6228–6237
ParkS. D.,
LeeS. N.,
ParkI. H.,
ChoiJ. S.,
JeongW. K.,
KimY.,
LeeH. S.2004; Isolation and characterization of transcriptional elements from Corynebacterium glutamicum . J Microbiol Biotechnol 14:789–795
SchäferA.,
TauchA.,
JägerW.,
KalinowskiJ.,
ThierbachG.,
PühlerA.1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene 145:69–73
SirisatthaS.,
MomoseY.,
KitagawaE.,
IwashashiH.2004; Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis. Water Res 38:61–70
von der OstenC. H.,
GioannettiC.,
SinskeyA. J.1989; Design of a defined medium for growth of Corynebacterium glutamicum in which citrate facilitates iron uptake. Biotechnol Lett 11:11–16