Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

Abstract

The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoteroperator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

DNA Repair and Genome Maintenance in Bacillus subtilis

“…In addition to these studies, the SOS regulon has also been investigated in other Gram-positive bacteria. The SOS responses in many other Gram-positive bacteria are also regulated by RecA and LexA, as expected (169,288). Overall, the number of genes and functions of genes controlled by this response differ considerably from organism to organism.…”

Positive transcriptional control of the pyridoxal phosphate biosynthesis genes pdxST by the MocR-type regulator PdxR of Corynebacterium glutamicum ATCC 13032

“…5a). Interestingly, motif 1 overlaps the previously detected DNA-binding site (SOS box) of the SOS response regulator LexA from C. glutamicum (Jochmann et al, 2009). An additional 12 bp sequence, termed motif 2 (AAAGTGGATCTA), showed similarity to motif 1 and motif 3 and was identified between the 235 promoter regions (Fig.…”

“…The peptide mass fingerprint of the purified PdxR protein was generated by MALDI-TOF MS, by using an Ultraflex mass spectrometer (Bruker Daltonics) and the MASCOT software. The LexA protein from C. glutamicum ATCC 13032 was purified as described previously (Jochmann et al, 2009).…”

“…Inactivation of the cg0897 (pdxR) gene by either random transposon mutagenesis with an IS6100-based mobile element (Mormann et al, 2006) or directed gene replacement (McHardy et al, 2003) resulted in vitamin B 6 auxotrophy of the respective mutant strains and indicated that the pdxST genes of C. glutamicum (cg0898 and cg0899) are probably under positive transcriptional control by PdxR. Furthermore, the level of pdxR gene expression was enhanced during the response of C. glutamicum to severe heat shock conditions (Barreiro et al, 2009), whereas the expression of the pdxST genes was slightly down-regulated in the lexA mutant C. glutamicum NJ2114 (Jochmann et al, 2009). In the latter study, an SOS box was detected upstream of the pdxST gene region and the specific binding of the SOS response regulator LexA was demonstrated in vitro by DNA band shift assays, integrating the pdxST genes into the SOS regulon of C. glutamicum.…”

A resposta SOS de Caulobacter crescentus e relações dos mecanismos de reparo com a progressão do ciclo celular.

“…Em R. sphaeroides, LexA é capaz de reprimir ou estimular a transcrição de recA, dependendo de sua concentração protéica . Na bactéria Gram positiva C. glutamicum, LexA parece ser capaz de induzir a expressão de pelo menos dois genes envolvidos em transporte e na biossíntese de vitamina B 6 (Jochmann et al, 2009 The prototypical cellular response to DNA damage in prokaryotes is the SOS response. Best characterized in Escherichia coli, it can be viewed as a global stress response system, which controls the expression of several genes in response to a wide variety of environmental challenges (reviewed in reference 24).…”