1887

Abstract

The surface (S)-layer gene region of the Gram-positive bacterium ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the ATCC 13032 chromosome was identified. This region includes , 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 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 ATCC 13032. Transfer of the cloned gene restored the PS2 phenotype of ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living cells by atomic force microscopy. Furthermore, the promoter of the 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 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 gene in resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of gene expression in .

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2006-04-01
2019-10-20
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