Surface array protein of Campylobacter fetus. Cloning and gene structure.
The high molecular mass (97-149 kDa) surface array proteins (SAP) of Campylobacter fetus are critical to virulence. We created a bank of 160,000 random 1.0-6.5-kilobase (kb) chromosomal DNA fragments of C. fetus strain 84-32 (23D) using lambda gt11. Screening this bank in Escherichia coli Y1090 with antibody raised against purified SAP permitted isolation and purification of a clone with a 4.0-kb insert. Subcloning this insert in the E. coli vector, pUC9, permitted expression of a protein of approximately 100 kDa, not fused with beta-galactoside or inducible by isopropyl-beta-D-thiogalactopyranoside. Digestion with restriction endonucleases, and construction of deletion mutations indicated that the gene extended over 2.8 kb, proceeding toward the start of the beta-galactosidase gene. Taking advantage of a unique PstI site at 1.7 kb, we subcloned PstI-EcoRI fragments in both orientations into M13 vectors, then generated and sequenced 48 deletion mutants. In the 3974-base insert, an open reading frame, beginning at nucleotide 24 and terminating at 2825, was found to encode a 933-amino acid polypeptide having a calculated molecular mass of 96,758 daltons. The first 20 amino acids exactly match those determined from amino-terminal sequencing, indicating that this protein is secreted without a leader sequence. The deduced amino acid composition matches that of the purified SAP. We identified a ribosomal binding site 9 bases upstream, and a putative transcription terminator (delta G = -12.4) 21 bases downstream. There is partial homology of primary and secondary structure with five other bacterial S-layer proteins.
Blaser
M J
MJ
Laboratory for Bacteriology and Immunology, Rockefeller University, New York, New York 10021.
Gotschlich
E C
EC
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins; D001483:Base Sequence; D002168:Campylobacter fetus / Q000235:genetics* / Q000472:pathogenicity; D003001:Cloning, Molecular; D015723:Gene Library; D005798:Genes, Bacterial; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013728:Terminator Regions, Genetic; D014774:Virulence / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C055764:S-layer proteins