Use of alkaline phosphatase as a reporter polypeptide to study the role of the subtilin leader segment and the SpaT transporter in the posttranslational modifications and secretion of subtilin in Bacillus subtilis 168.
The subtilin leader segment of presubtilin was fused to alkaline phosphatase (AP), which was used as a reporter polypeptide to study the role of the subtilin leader segment in posttranslational modifications during the conversion of presubtilin to subtilin and in the translocation of presubtilin from the cytoplasm of Bacillus subtilis 168 to the extracellular medium. It was observed that the subtilin leader segment could be utilized by a wild-type transporter, but secretion was enhanced if the subtilin transporter was available. The subtilin leader was not cleaved away from the AP component of the precursor until the precursor had been transported to the cell wall, and none of the AP was released into the medium until after cleavage had occurred. The role of SpaT, which is an ABC transporter that has been implicated in subtilin secretion, was explored by making a large in-frame deletion from the central region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deletion disrupted proteolytic cleavage of the subtilin leader segment and release of the AP reporter into the medium. The AP that was secreted by means of the subtilin leader segment had not undergone any posttranslational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.
Izaguirre
G
G
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Hansen
J N
JN
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins; D001430:Bacteriocins; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D008565:Membrane Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C104627:SpaT protein, Bacillus subtilis; D000469:Alkaline Phosphatase; C002836:subtilin