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

• Published in: Microbiology (Society for General Microbiology) Vol. 155; no. 5; pp. 1459 - 1477
• Main Authors: Jochmann, Nina, Kurze, Anna-Katharina, Czaja, Lisa F, Brinkrolf, Karina, Brune, Iris, Huser, Andrea T, Hansmeier, Nicole, Puhler, Alfred, Borovok, Ilya, Tauch, Andreas
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01-05-2009; Society for General Microbiology
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Base Sequence; Binding Sites; Biological and medical sciences; Conserved Sequence; Corynebacterium glutamicum; Corynebacterium glutamicum - chemistry; Corynebacterium glutamicum - genetics; Corynebacterium glutamicum - metabolism; Electrophoretic Mobility Shift Assay; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Microbiology; Miscellaneous; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Regulon; Sequence Deletion; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Transcription, Genetic; Bacteria; Genetics; Actinomycetes; Corynebacteriaceae; In vitro; Corynebacterium glutamicum
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.025841-0

1 Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 27, D-33615 Bielefeld, Germany
2 International NRW Graduate School in Bioinformatics and Genome Research, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany
3 Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA
4 George S. Wise Faculty of Life Sciences, Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel
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 promoter–operator 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.
Correspondence Andreas Tauch Andreas.Tauch{at}genetik.uni-bielefeld.de
Abbreviations: AFM, atomic force microscopy; Cy3, indocarbocyanine; HMM, hidden Markov model; PLP, pyridoxal 5'-phosphate; RACE, rapid amplification of cDNA ends
Present address: Evonik Degussa GmbH, D-33790 Halle (Westf.), Germany.
A supplementary table, listing oligonucleotides used in this study, is available with the online version of this paper.
The microarray hybridization data discussed in this paper have been deposited in the CoryneRegNet database ( http://www.coryneregnet.de/ ) with identifiers ‘delta_lexa’ and ‘mitomycin c’, and can be downloaded for further analysis by using SOAP-based web services (Baumbach & Apeltsin, 2008).