Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47: complete nucleotide sequence of the outer wall protein gene

Abstract

BaciUus brevis 47 contains two cell wall proteins termed the outer wall protein (OWP) and middle wall protein (MWP), each of which forms hexagonal arrays in the cell wall. A 6-kilobase Bglll-Bcll fragment of B. brevis 47 DNA cloned into BaciUus subtilis with a derivative of pHWl as a vector directed the synthesis of a polypeptide, with almost the same molecular weight as the authentic OWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was specifically recognized by the anti-OWP antibody. Nucleotide sequence analysis of the subfragment revealed that it contains two open reading frames in tandem.The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the MWP, and the downstream open reading frame corresponds to the entire translational portion of the OWP. The latter encodes a secretory precursor of the OWP, consisting of a total of 1,004 amino acid residues with a signal peptide of 24 amino acid residues at its amino-terminal end. Furthermore, analysis of transcripts in B. brevis 47 suggests that the MWP and OWP genes, in that order, constitute a cotranscriptional unit and that the major promoter shared by the two genes is located upstream of the MWP gene.

Molecular characterization of the Bacillus anthracis main S‐layer component: evidence that it is the major cell‐associated antigen

“…Clostridium perfringens (Takumi et a/., 1991), the S-layer is composed of more than one protein. In other organisms, such as Aquaspirillum serpens (Kist and Murray, 1984), Clostridium difficile (Kawata et a/., 1984) and B. brevis (Tsuboi et a/., 1986), there appear to be two superimposed S-layers. In other bacteria, including Bacillus stearothermophilus and Campylobacter fetus (Tummuru and Blaser, 1993), more than one S-layer can appear sequentially.…”

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy

“…This particularly applies to subunit-subunit interactions, to regions exposed to the environment, and to regions interacting with the underlying cell envelope components. Such a description is becoming possible for several species whose S-layer genes have been cloned and sequenced and for which the possibil-ity for high-resolution image analysis exists (6,7,17,18,30,31). C. crescentus is such a species.…”

S-layers of Bacillus species

“…The gene encoding the non-glycosylated, hexagonally arranged S-layer of B. stearothermophilus PV72 was cloned and sequenced by Kuen et al (1994). These authors showed sequence comparisons with other signal sequences and found a high degree of homology within signal sequences of extracellular proteins in Bacillus species, such as alkaline phosphatase and neutral protease from B. amyloliquefaciens (Vasantha et al, 1984), as well as the signal sequence for B. sphaericus gene 125 (Bowditch et al, 1989), and the outer-wall protein gene for B. brevis (Tsuboi et al, 1986). Peptide mapping and N-terminal sequencing results strongly indicated that the S-layer protein of B. stearotherrnophilus NRS 2004/3a and two induced variant strains are encoded by different genes and are not derived from a universal precursor form .…”

“…A portion of each of the OWP and MWP (middle-wall protein) genes of B. brevis 47 was cloned in E. coli by an immunological procedure (Tsukagoshi et al, 1984). Analysis of transcripts in B. brevis 47 revealed that the genes for the MWP and OWP constitute a cotranscriptional unit and they are transcribed from multiply and tandemly arranged promoters located upstream of the MWP gene [ c w p (cell-wall protein gene) operon] (Yamagata et al, 1987;Tsuboi et al, 1986). After analysis of the DNA sequence of the promoter region (Yamagata et al, 1987), the complete nucletide sequence of the cwp operon was determined (Tsuboi et al, 1988).…”

“…The DNA fragments of the protein-producing B. brevis 47 were first cloned into Escherichia coli and B. subtilis (Yamagata et al, 1987;Tsukagoshi et al, 1984). The complete nucleotide sequence of the OWP (outer-wall protein) gene of B. brevis 47 was determined by Tsuboi et al (1986). A portion of each of the OWP and MWP (middle-wall protein) genes of B. brevis 47 was cloned in E. coli by an immunological procedure (Tsukagoshi et al, 1984).…”