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

Nicole Hansmeier, Andreas Albersmeier, Andreas Tauch, Thomas Damberg, Robert Ros, Dario Anselmetti, Alfred Pühler, Jörn Kalinowski

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The surface (S)-layer gene region of the Gram-positive bacterium Colynebacterium 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.

Original languageEnglish (US)
Pages (from-to)923-935
Number of pages13
JournalMicrobiology
Volume152
Issue number4
DOIs
StatePublished - Apr 2006
Externally publishedYes

Fingerprint

Corynebacterium glutamicum
Genomic Islands
Genes
DNA
Polymerase Chain Reaction
Proteins
Peptide Mapping
Atomic Force Microscopy
Nucleic Acid Repetitive Sequences
Protein Subunits
Gram-Positive Bacteria
Genetic Promoter Regions
Protein Binding
Genetic Recombination
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Lasers
Complementary DNA
Clone Cells
Chromosomes

ASJC Scopus subject areas

  • Microbiology

Cite this

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. / Hansmeier, Nicole; Albersmeier, Andreas; Tauch, Andreas; Damberg, Thomas; Ros, Robert; Anselmetti, Dario; Pühler, Alfred; Kalinowski, Jörn.

In: Microbiology, Vol. 152, No. 4, 04.2006, p. 923-935.

Research output: Contribution to journalArticle

Hansmeier, Nicole ; Albersmeier, Andreas ; Tauch, Andreas ; Damberg, Thomas ; Ros, Robert ; Anselmetti, Dario ; Pühler, Alfred ; Kalinowski, Jörn. / 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. In: Microbiology. 2006 ; Vol. 152, No. 4. pp. 923-935.
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abstract = "The surface (S)-layer gene region of the Gram-positive bacterium Colynebacterium 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.",
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T1 - 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

AU - Hansmeier, Nicole

AU - Albersmeier, Andreas

AU - Tauch, Andreas

AU - Damberg, Thomas

AU - Ros, Robert

AU - Anselmetti, Dario

AU - Pühler, Alfred

AU - Kalinowski, Jörn

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AB - The surface (S)-layer gene region of the Gram-positive bacterium Colynebacterium 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.

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