Alterations in the carboxy-terminal half of cloacin destabilize the protein and prevent its export by Escherichia coli.
Several overlapping carboxy-terminal and internal deletions were constructed in the cloacin structural gene. The expression, the binding of the cloacin DF13 immunity protein and the release into the culture medium of the mutant cloacin polypeptides were studied by immunoblotting and ELISAs. Minor alterations at the carboxy-terminal end of the cloacin did not affect protein expression, stability or release to a large extent, but larger carboxy-terminal deletions strongly destabilized the protein and no release was observed. The removal of a particular region within the carboxy-terminal portion of cloacin strongly destabilized the polypeptide and made it a target for proteolytic degradation. Binding of immunity protein did not affect stability and release of the mutant polypeptides. By using immunoelectron microscopy, the polypeptides that were not exported were located in the cytoplasm of producing cells. Large aggregates of these mutant polypeptides were not observed in the cytoplasm: the polypeptides were present in a soluble form.
van Putten
A J
AJ
Department of Molecular Microbilogy, Vrije Universiteit, Amsterdam.
Stegehuis
F
F
van Bergen en Henegouwen
P M
PM
De Graaf
F K
FK
Oudega
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001430:Bacteriocins / Q000235:genetics*; D002989:Cloacin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping
D001426:Bacterial Proteins; D001430:Bacteriocins; D029968:Escherichia coli Proteins; C027355:cloacin DF13 protein, E coli; D002989:Cloacin