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Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47:
Abstract:
Bacillus 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 BglII-BclI fragment of B. brevis 47 DNA cloned into Bacillus subtilis with a derivative of pHW1 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. Futhermore, 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.
Insights
Bacillus brevis 47 outer wall protein (OWP) and middle wall protein (MWP) genes are cotranscribed. DNA cloning and sequencing identified the OWP gene encoding a secretory precursor and revealed a shared promoter upstream of the MWP gene.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Bacillus brevis 47 possesses two distinct cell wall proteins, outer wall protein (OWP) and middle wall protein (MWP).
- Both OWP and MWP form hexagonal arrays, contributing to the structural integrity of the bacterial cell wall.
Purpose of the Study:
- To investigate the genetic organization and expression of the OWP and MWP genes in Bacillus brevis 47.
- To identify the DNA sequences responsible for the synthesis of OWP and MWP.
Main Methods:
- Cloning of a DNA fragment from B. brevis 47 into Bacillus subtilis using a pHW1 vector derivative.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess polypeptide molecular weight.
- Antibody recognition assays using anti-OWP antibodies.
- Nucleotide sequence analysis of cloned DNA fragments.
- Transcript analysis in B. brevis 47.
Main Results:
- A cloned DNA fragment directed the synthesis of a polypeptide recognized by anti-OWP antibodies, indicating successful expression of the OWP gene.
- Sequence analysis revealed two tandem open reading frames, encoding the C-terminal portion of MWP and the full OWP precursor.
- The OWP precursor consists of 1,004 amino acid residues, including a 24-amino acid signal peptide.
- Transcript analysis indicated that the MWP and OWP genes form a cotranscriptional unit with a shared promoter upstream of the MWP gene.
Conclusions:
- The OWP and MWP genes in Bacillus brevis 47 are organized in tandem and likely cotranscribed from a promoter located upstream of the MWP gene.
- The identified DNA sequence encodes a secretory precursor of the OWP, providing insights into its synthesis and localization.