Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum.
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 63,000 Da, corresponding presumably to the mature 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.
Peyret
J L
JL
Laboratoire de Biologie Moléculaire des Coryné-bactéries, URA D1354 CNRS et GDR 961, Université Paris-Sud, Orsay, France.
Bayan
N
N
Joliff
G
G
Gulik-Krzywicki
T
T
Mathieu
L
L
Schechter
E
E
Leblon
G
G
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D005613:Freeze Etching; D005798:Genes, Bacterial; D007526:Isoelectric Point; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins