Complete nucleotide sequence and comparative genomic analysis of microcin B17 plasmid pMccB17

Abstract

We present a comprehensive sequence and bioinformatic analysis of the prototypical microcin plasmid, pMccb17, which includes a definitive sequence for the microcin operon, mcb. Microcin B17 (MccB17) is a ribosomally synthesized and posttranslationally modified peptide produced by Escherichia coli. It inhibits bacterial DNA gyrase similarly to quinolone antibiotics. The mcb operon, which consists of seven genes encoding biosynthetic and immunity/export functions, was originally located on the low copy number IncFII plasmid pMccB17 in the Escherichia coli strain LP17. It was later transferred to E. coli K-12 through conjugation. In this study, the plasmid was extracted from the E. coli K-12 strain RYC1000 [pMccB17] and sequenced twice using an Illumina short-read method. The first sequencing was conducted with the host bacterial chromosome, and the plasmid DNA was then purified and sequenced separately. After assembly into a single contig, polymerase chain reaction primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete circular DNA sequence is 69,190 bp long and includes 81 predicted genes. These genes were initially identified by Prokka and subsequently manually reannotated using BLAST. The plasmid was assigned to the F2:A-:B- replicon type with a MOBF12 group conjugation system. A comparison with other IncFII plasmids revealed a large proportion of shared genes, particularly in the conjugative plasmid backbone. However, unlike many contemporary IncFII plasmids, pMccB17 lacks transposable elements and antibiotic resistance genes. In addition to the mcb operon, this plasmid carries 25 genes of unknown function.

Keywords: Enterobacteriaceae; genome; microcin; plasmid.

Figure 1

Circular Map of pMccB17. The outer ring shows the size of the plasmid, each tick representing 4 kb. The microcin operon is shown in brown, plasmid maintenance systems are shown in yellow, the replicon is shown in pink the conjugative transfer system is represented in blue, hypothetical proteins are shown in turquoise, origins of replication are in green and the 149 bp direct repeats are shown in red. Analysis based on the complete sequence as submitted to Genbank (ON989342). Diagram generated using DNAplotter (Carver et al., 2009).

Figure A1

Direct repeat sequence from pMccB17. This sequence occurs twice in the pMccB17 genome. (a) The 149 bp repeated sequence. The two arms of a large perfect palindrome at the center of the sequence have been underlined. (b) Predicted secondary structure formed by RNA encoded by this sequence (initial ΔG = −67.80). A highly similar structure was predicted for ssDNA (dG = −35.09). These structures were predicted using mfold version 3.6, with default parameters, as implemented by the UNAFold Web Server (Zuker, ; www.unafold.org).

Figure A2

Phylogenetic analysis of ParB family proteins. Pbf proteins are highlighted in red. The clades are named in this order: name of the ParB family protein, two letters representing the organism it is found, plasmid it is found in (only in Pbf), and plasmid incompatibility group (only in Pbf). EC = Escherichia coli; SE = Salmonella enterica; S = Salmonella; PA = Pseudomonas aeruginosa; Ssp. = Streptomyces sp.; ED = Enterococcus durans; TT = Thermus thermophilus; BS = Bacillus subtilis; EPp1 = Escherichia phage P1; SF = Shigella flexneri; CB = Coxiella burnetii; DR = Deinococcus radiodurans; VC = Vibrio cholerae; NM = Neisseria meningitidis; PP = Pseudomonas putida. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel (L + G) model. The tree with the highest log likelihood (−5208.41) is shown. The percentage of trees in which the associated taxa clustered together is shown below the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor‐Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories ( + G, parameter = 3.2901)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions with less than 95% site coverage were eliminated, that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 169 positions in the final data set. Evolutionary analyses were conducted in MEGA X.