Cloning and expression of genes responsible for altered penicillin-binding proteins 3a and 3b in Haemophilus influenzae.
A Haemophilus influenzae strain (T-1,3) possessing clinical beta-lactam resistance due to altered penicillin-binding protein 3 was used to construct a recombinant cosmid gene bank in Escherichia coli. Three of the recombinant cosmids were capable of transforming a susceptible H. influenzae strain (Rdnov) simultaneously to moxalactam resistance and altered the binding of penicillin-binding proteins 3a and 3b to [35S]penicillin G. Restriction endonuclease mapping of one of the recombinant cosmids, pLB100, was performed to facilitate subsequent subcloning of the gene(s) responsible for the altered penicillin-binding protein 3 (a and b) binding phenotype. Subcloning of individual fragments derived from pLB100 indicated that two adjacent fragments of DNA were both capable of transforming a susceptible Haemophilus strain to moxalactam resistance and altered penicillin-binding protein 3 binding. Expression of plasmid-coded proteins in minicells indicated that one fragment coded for a major 55,000-molecular-weight polypeptide and that the second contained a C-terminal coding region that expressed a 28,000-molecular-weight polypeptide when fused to the N-terminal region of the tetracycline resistance gene. Initial attempts at labeling the plasmid-coded proteins expressed in minicells with [35S]penicillin G were unsuccessful.
D000217:Acyltransferases / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D009070:Moxalactam / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G / Q000378:metabolism / Q000494:pharmacology; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000096:biosynthesis / Q000235:genetics*; D010878:Piperacillin / Q000494:pharmacology; D010957:Plasmids; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G; D009070:Moxalactam; D010878:Piperacillin