Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance

The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.     

From resistance to stimulation: the evolution of a virus in the presence of a dominant lethal inhibitory scaffolding protein

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks φX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.     

The population biology of bacterial plasmids: a hidden Markov model approach

Horizontal plasmid transfer plays a key role in bacterial adaptation. In harsh environments, bacterial populations adapt by sampling genetic material from a horizontal gene pool through self-transmissible plasmids, and that allows persistence of these mobile genetic elements. In the absence of selection for plasmid-encoded traits it is not well understood if and how plasmids persist in bacterial communities. Here we present three models of the dynamics of plasmid persistence in the absence of selection. The models consider plasmid loss (segregation), plasmid cost, conjugative plasmid transfer, and observation error. Also, we present a stochastic model in which the relative fitness of the plasmid-free cells was modeled as a random variable affected by an environmental process using a hidden Markov model (HMM). Extensive simulations showed that the estimates from the proposed model are nearly unbiased. Likelihood-ratio tests showed that the dynamics of plasmid persistence are strongly dependent on the host type. Accounting for stochasticity was necessary to explain four of seven time-series data sets, thus confirming that plasmid persistence needs to be understood as a stochastic process. This work can be viewed as a conceptual starting point under which new plasmid persistence hypotheses can be tested.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Expression of the gene for tetracycline resistance of plasmids R6 and RP4 in bacteria of the family Enterobacteriaceae]

It was found that manifestation of the tetracycline resistance gene depended on the type of the plasmid containing the gene. The tetracycline resistance gene was subject to less repression in plasmid R6 than in plasmid RP4. Sensitivity of the initial plasmid-free bacteria varied within lower dose ranges than that of the plasmid-carrying strains. Regulation of the tetracycline resistance gene manifestation in the given plasmid may change in different bacterial hosts, i.e. in different cytoplasmic environment at different gene background.     

Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B.

We use a mathematical model to analyze the dynamics of loss of nonconjugative pACYC184 from populations of Escherichia coli B in glucose-limited continuous culture. This model incorporates both plasmid segregation and selection against plasmid carriage. It is concluded that there is intense selection against plasmid carriage (s = 0.3 per culture generation), which amplifies the frequency of segregants arising de novo.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

In vivo andin vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate ofEscherichia coli

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.     

The Yersinia enterocolitica pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (相似文献   

Epistatic effects of promoter and repressor functions of the Tn10 tetracycline-resistance operon on the fitness of Escherichia coli

We have been studying the effects of expression of plasmid-borne, Tn10-encoded, tetracycline resistance on the fitness of Escherichia coli K12. We previously demonstrated large reductions in fitness resulting from induced or constitutive expression of the resistance protein; however, any residual expression by the repressed operon was so slight that possession of an inducible resistance function imposed essentially no burden in the absence of antibiotic. Here, we demonstrate two distinct disadvantages for inducible genotypes relative to isogenic constitutive constructs. During the transition from antibiotic-free to antibiotic-containing media, the inducible genotype experiences a longer lag phase prior to growth. In the sustained presence of antibiotic, full induction of the resistance function in the inducible genotype is prevented by the continued action of its repressor. However, these disadvantages may be reduced by increasing the strength of the promoter for the resistance gene in the inducible genotype. Simultaneous consideration of the mode of gene regulation (i.e. constitutive or inducible) and the strength of the resistance-gene promoter (i.e. maximum level of expression) indicates an adaptive landscape with very strong epistasis and, perhaps, multiple fitness peaks.     

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.     

Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in pseudomonads

Environmental conditions under which fitness tradeoffs of plasmid carriage are balanced to facilitate plasmid persistence remain elusive. Periodic selection for plasmid-encoded traits due to the spatial and temporal variation typical in most natural environments (such as soil particles, plant leaf and root surfaces, gut linings, and the skin) may play a role. However, quantification of selection pressures and their effects is difficult at a scale relevant to the bacterium in situ. The present work describes a novel experimental system for such fine-scale quantification, with conditions designed to mimic the mosaic of spatially variable selection pressures present in natural surface environments. The effects of uniform and spatially heterogeneous mercuric chloride (HgCl(2)) on the dynamics of a model community of plasmid-carrying, mercury-resistant (Hg(r)) and plasmid-free, mercury-sensitive (Hg(s)) pseudomonads were compared. Hg resulted in an increase in the surface area occupied by, and therefore an increase in the fitness of, Hg(r) bacteria relative to Hg(s) bacteria. Uniform and heterogeneous Hg distributions were demonstrated to result in different community structures by epifluorescence microscopy, with heterogeneous Hg producing spatially variable selection landscapes. The effects of heterogeneous Hg were only apparent at scales of a few hundred micrometers, emphasizing the importance of using appropriate analysis methods to detect effects of environmental heterogeneity on community dynamics. Heterogeneous Hg resulted in negative frequency-dependent selection for Hg(r) cells, suggesting that sporadic selection may facilitate the discontinuous distribution of plasmids through host populations in complex, structured environments.     

Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry

A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.     

Effects of carriage and expression of the Tn10 tetracycline-resistance operon on the fitness of Escherichia coli K12

We have been examining the consequences of alternative modes of regulation of plasmid-borne, Tn10-encoded tetracycline resistance for the fitness of Escherichia coli. In a tetracycline-free environment, we measured the effects on fitness that were caused by (1) maximally induced expression of the resistance operon, (2) low-level constitutive expression of the resistance protein, (3) residual expression of the repressed resistance operon, (4) carriage of the resistance operon, (5) the remainder of the plasmid genome, and (6) hyperexpression of the repressor protein. We observed large reductions in fitness that were associated with induction and with constitutive expression of the tetracycline-resistance protein, but there was no discernible effect of hyperexpression of the repressor protein. We also observed a small reduction in fitness associated with the remainder of the plasmid genome. However, any reductions in fitness that were caused by residual expression and by carriage of the repressed operon were not more than 0.3%. We conclude that tight gene regulation has eliminated antagonistic pleiotropic effects of the resistance gene on fitness, so that possession of an inducible Tn10-encoded tetracycline-resistance operon imposes essentially no burden in the absence of antibiotic.