CHARACTERIZATION OF THE CSPB GENE ENCODING PS2, AN ORDERED SURFACE-LAYER PROTEIN IN CORYNEBACTERIUM-GLUTAMICUM

被引:91
作者
PEYRET, JL
BAYAN, N
JOLIFF, G
GULIKKRZYWICKI, T
MATHIEU, L
SHECHTER, E
LEBLON, G
机构
[1] UNIV PARIS 11,BIOL MOLEC CORYNEBACTERIES LAB,CNRS,URA D1354,BAT 409,F-91405 ORSAY,FRANCE
[2] UNIV PARIS 11,GDR 961,F-91405 ORSAY,FRANCE
[3] UNIV PARIS 11,BIOMEMBRANE LAB,CNRS,URA 1116,F-91405 ORSAY,FRANCE
[4] CTR ORSAN RECH BIOTECHNOL,F-91953 COURTABOEUF,FRANCE
[5] CNRS,CTR GENET MOLEC,F-91190 GIF SUR YVETTE,FRANCE
关键词
D O I
10.1111/j.1365-2958.1993.tb01672.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
PS2 is one of two major proteins detected in the culture media of various CorYnebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63 000Da, corresponding presumably to the nature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65 000, 68 000 and 72 000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510-amino-acid polypeptide had a calculated molecular mass of 55 426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a N-terminal segment of 30-amino-acid residues reminiscent of eukaryotic and prokaryotic signal peptides, and a hydrophobic domain of 21 residues near the C-terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface-layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze-etching electron microscopy performed on the wild-type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S-layer) which is in very close contact with other cell-wall components. These structures are absent from the cspB-disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the S-layer protein is the product of the cspB gene.
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页码:97 / 109
页数:13
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