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Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1␤ replicon-specific sequences detected in total community DNA. The isolation of IncP-1␤ plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1␤ plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1␤ plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRT⌬P probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1␤ plasmids R751 and pB8. However, this is the first example of an IncP-1␤ plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules.

BackgroundUnderstanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin.Methodology/Principal FindingsHigh throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates.Conclusions/SignificancePlasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

Plasmid Rms149, the archetype of Pseudomonas plasmid incompatibility group IncP-6, was identified in Pseudomonas aeruginosa as an agent conferring resistance to streptomycin, sulfanilamide, gentamicin, and carbenicillin in 1975. It has been classed as a broad-host-range plasmid due to its ability to replicate in both Escherichia coli (where it is designated IncG) and Pseudomonas species, although both species are ␥-proteobacteria. To provide reference information on this Inc group, we have determined the complete sequence of Rms149 and found that, although the genome comprises 57,121 bp, it is essentially a small mobilizable plasmid carrying multiple mobile elements, which make up 79% (>45 kb) of its genome. A replicon has been identified which encodes a single polypeptide with moderate identity to other replication proteins. The region encoding this protein can replicate in Pseudomonas putida and E. coli. This sequence is directly downstream of a putative partitioning region highly similar to that of pRA2. A functional IncQ-type mobilization region is also present. Thus, the backbone appears to be a novel combination of modules already identified in other plasmid systems. Analysis of the segments that fall outside this core of stable inheritance and transfer functions show that this plasmid has been subject to multiple insertion events and that the plasmid appears to carry a considerable load of DNA that no longer should be phenotypically advantageous. The plasmid therefore functions not just as a vehicle for spread of selective traits but also as a store for DNA that is not currently under selection.The key features of a plasmid are the ability to replicate autonomously and to be maintained in a cell lineage without a high rate of segregational loss. An optional but frequently encountered property is the ability to transfer or to be mobilized from one bacterium to another. From these properties it follows that genes that become associated with a plasmid may spread rapidly from one genetic background to another. Some plasmids appear to consist of nothing more than functions that confer the above core abilities, and these have been termed cryptic plasmids.To succeed as such a selfish element, a plasmid must be stable and confer minimal burden on its host, or at least overcome competitive losses by horizontal transfer (42). Conversely, a plasmid carrying genes that promote the growth of its host, relative to competitor bacteria, will benefit by increased propagation. However, since maintenance of a plasmid is generally found to place a metabolic or phenotypic load on the cell, universally useful genes will be selectively reacquired by the chromosome over evolutionary time. Therefore, successful plasmids typically carry payload genes, favorable in some environments but not others (12), or genes with a transient plasmid association that are spreading through a microbial community. In passing through different strains or species, plasmids are exposed to different genetic contexts and provide the opportunity for comb...

SummaryThe incC and korB genes of IncP-1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immuno¯uorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP-1 plasmids. When maintained by the low-copy-number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an ef®cient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP-1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one-and three-quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern ®ts with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning.

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2?1610 "4 to 1?75610 "5 were obtained for five of the nine plasmids -the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1b subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1c. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.