Evolution of satellite plasmids can prolong the maintenance of newly acquired accessory genes in bacteria

Transmissible plasmids spread genes encoding antibiotic resistance and other traits to new bacterial species. Here we report that laboratory populations of Escherichia coli with a newly acquired IncQ plasmid often evolve ‘satellite plasmids’ with deletions of accessory genes and genes required for plasmid replication. Satellite plasmids are molecular parasites: their presence reduces the copy number of the full-length plasmid on which they rely for their continued replication. Cells with satellite plasmids gain an immediate fitness advantage from reducing burdensome expression of accessory genes. Yet, they maintain copies of these genes and the complete plasmid, which potentially enables them to benefit from and transmit the traits they encode in the future. Evolution of satellite plasmids is transient. Cells that entirely lose accessory gene function or plasmid mobility dominate in the long run. Satellite plasmids also evolve in Snodgrassella alvi colonizing the honey bee gut, suggesting that this mechanism may broadly contribute to the importance of IncQ plasmids as agents of bacterial gene transfer in nature.

Evolution of satellite plasmids can stabilize the maintenance of newly acquired accessory genes in bacteria

Plasmids play a principal role in the spread of antibiotic resistance and other traits by horizontal gene transfer in bacteria. However, newly acquired plasmids generally impose a fitness burden on a cell, and they are lost from a population rapidly if there is not selection to maintain a unique function encoded on the plasmid. Mutations that ameliorate this fitness cost can sometimes eventually stabilize a plasmid in a new host, but they typically do so by inactivating some of its novel accessory genes. In this study, we identified an additional evolutionary pathway that can prolong the maintenance of newly acquired genes encoded on a plasmid. We discovered that propagation of an RSF1010-based IncQ plasmid inEscherichia colioften generated ‘satellite plasmids’ with spontaneous deletions of accessory genes and genes required for plasmid replication. These smaller plasmid variants are nonautonomous genetic parasites. Their presence in a cell drives down the copy number of full-length plasmids, which reduces the burden from the accessory genes without eliminating them entirely. The evolution of satellite plasmids may be favored relative to other plasmid fates because they give a more immediate fitness advantage to a cell’s progeny and because the organization of IncQ plasmids makes them particularly prone to certain deletions during replication. Satellite plasmids also evolved inSnodgrassella alvicolonizing the honey bee gut, suggesting that this mechanism may broadly contribute to the importance of IncQ plasmids as agents of bacterial gene transfer in nature.Significance StatementPlasmids are multicopy DNA elements found in bacteria that replicate independently of a cell’s chromosome. The spread of plasmids carrying antibiotic-resistance genes to new bacterial pathogens is a challenge for treating life-threatening infections. Often plasmids or their accessory genes encoding unique functions are lost soon after transfer into a new cell because they impose a fitness burden. We report that a family of transmissible plasmids can rapidly evolve ‘satellite plasmids’ that replicate as genetic parasites of the original plasmid. Satellite plasmid formation reduces the burden from the newly acquired genes, which may enable them to survive intact for longer after transfer into a new cell and thereby contribute to the spread of antibiotic resistance and other traits within bacterial populations.

Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype

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 2.1×10−4 to 1.75×10−5 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-1β 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-1γ. 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.

Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block vir-Dependent Transfer of IncQ Plasmids between Agrobacterium Cells and the Establishment of IncQ Plasmids in Plant Cells

ABSTRACT The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

ABSTRACT The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl2), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cdr or Nir density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Nir transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

Functional Subsets of the VirB Type IV Transport Complex Proteins Involved in the Capacity of Agrobacterium tumefaciens To Serve as a Recipient in virB-Mediated Conjugal Transfer of Plasmid RSF1010

ABSTRACT The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer.

Comparative Biology of IncQ and IncQ-Like Plasmids

SUMMARY Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.