Abstract

Researchers are actively searching for new antibiotics or alternatives to antibiotics to combat the global challenge of microbial resistance. One group of alternative antimicrobials that have been intensively studied are the microcins. Microcin B17 (MccB17) is a ribosomally synthesized and post-translationally modified peptide (RiPP) that targets DNA gyrase subunit B. MccB17 is encoded by 7 genes arranged in an operon (mcb operon) carried on a large conjugative plasmid, pMccB17, that was originally found in E. coli LP17.

This work provided complete DNA sequence and in-depth bioinformatic analysis of the 69 kb circular pMccB17 plasmid and confirmed its previous assignment to the IncFII family. Replicon typing using the FAB formula placed pMccB17 into F2:A-:B- and it was assigned to the MOBF12 group. pMccB17 has an average GC content of 51% and 81 predicted coding sequences, 30 of which encode conjugation proteins and as previously suggested, it has no antibiotic resistance genes. Plasmid-encoded Pbf protein, homologous to proteins involved in plasmid or chromosome partitioning, was of particular interest due to its conservation in other IncF plasmids and its unknown function. Sequence variations in mcbB, mcbD and mcbF between the historic sequences of mcb operon (GenBank accession numbers M24253 and X07875) in comparison to the mcb operon from this work were noted which account for 2 amino acids changes in the encoded proteins of McbB and McbD, and 17 amino acids changes in the encoded protein of McbF.

Homologues of the mcb operon genes were discovered in the genome of the plant pathogen Erwinia persicina NBRC 102418. In previous work, the microcin operon from E. persicina (emo operon) has been PCR amplified with and without an extra ORF found downstream of its operon, in addition to the predicted upstream promoter region, and cloned into the E. coli pUC 18 and pUC19 plasmid vectors. When transformed into E. coli DH5α for expression, the pUC19-derived constructs showed a slightly higher level of microcin production (10%) which could be due to the orientation of the lac promoter in the vectors. The presence of the extra ORF was found not to affect microcin production. Bioassay experiments carried out to characterise the Erwinia persicina microcin (EMB17) showed that it is maximally produced during the stationary phase of growth. EMB17 has a narrow activity range being able to inhibit the growth of some proteobacteria such as Pseudomonas aeruginosa and Klebsiella pneumonia, but not the growth of any Gram-positive bacteria tested. Two peaks having the same mass of 1088.9 m/z were picked up by mass spectrometer during the analysis of EMB17, purification parameters were set around this mass using preparative HPLC-MS. When tested, it was confirmed that the purified product retained its antimicrobial activity.

The mcbA and mcbB genes from emo and mcb operons were individually deleted from these operons and cloned into inducible expression vectors for expression in trans. The single cloned gene was then paired with the remainder of the operon complementarily to make up cognate and non-cognate microcin operons in trans. Comparison of bioassay results from cognate and non-cognate reassembled operon indicated that the protein-protein interaction between the promicrocin and the synthase is not species-specific. The resulting microcin was active against the sensitive E. coli BL21 strain. Cis operon arrangement favours more microcin production in mcb operon, while the trans arrangement favours microcin production in emo operon. This molecular promiscuity may facilitate the engineering of mutant microcins with altered chemistry and potentially improved or novel antimicrobial properties.