Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17

Abstract

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.

The Highly Conserved TldD and TldE Proteins of Escherichia coli Are Involved in Microcin B17 Processing and in CcdA Degradation

“…1). It is likely that this form is pro-MccB17, as previously suggested (42). This form is either not secreted or not competent for uptake by MccB17-sensitive bacteria (Fig.…”

“…Rodriguez-Sainz et al proposed that a tldE mutant strain (described as a pmbA1 mutant) is deficient in maturation and secretion of MccB17 (42). Using a recA::lacZ fusion, they observed strong induction of the SOS system in the pmbA1 strain carrying a plasmid encoding MccB17.…”

“…The roles of the groELS, tldD, and tldE genes in CcdB resistance remain unclear, but Murayama and colleagues suggested that the corresponding proteins could enhance the CcdB-gyrase interaction (39). The tldE gene is the same gene as pmbA, previously identified by Rodriguez-Sainz and colleagues as required for the maturation and secretion of MccB17 (42).…”

“…In this paper we present in vivo data showing that TldD, as was previously described for TldE (PmbA), is involved in MccB17 secretion (42). The pro-MccB17 accumulates in ⌬tldD, ⌬tldE, and ⌬tldD ⌬tldE mutants, showing that both proteins are essential to removal of the 26-amino-acid leader peptide of pro-MccB17.…”

“…The product of the last gene in the mccB17 operon, mcbG, confers immunity to MccB17 by an unknown mechanism. A chromosomal gene called pmbA (or tldE) has been implicated in the maturation and secretion of MccB17 (42). The PmbA protein might be either the peptidase responsible for maturation or a chaperone promoting the interaction of pro-MccB17 with the specific McbEF pump, where cleavage would take place.…”