The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032.
The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5.97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2(-) phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5' rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.
Institut für Genomforschung, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.|Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Nicole
N
Hansmeier
Institut für Genomforschung, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.|Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Andreas
A
Albersmeier
Institut für Genomforschung, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Andreas
A
Tauch
Lehrstuhl für Experimentelle Biophysik und Angewandte Nanowissenschaften, Fakultät für Physik, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Thomas
T
Damberg
Lehrstuhl für Experimentelle Biophysik und Angewandte Nanowissenschaften, Fakultät für Physik, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Robert
R
Ros
Lehrstuhl für Experimentelle Biophysik und Angewandte Nanowissenschaften, Fakultät für Physik, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Dario
D
Anselmetti
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Alfred
A
Pühler
Institut für Genomforschung, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Jörn
J
Kalinowski
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D044404:Genomic Islands; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020543:Proteome / Q000032:analysis; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D026801:Synteny; D020079:Terminal Repeat Sequences; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020543:Proteome; D012097:Repressor Proteins; D015534:Trans-Activators; C077188:cold-shock protein CspB, Bacteria; C064778:LuxR autoinducer binding proteins