Efficient transfer of conjugative plasmids by multipoint inoculation and some observations on host range and prevalence of R plasmids.

The conjugational transfer of R plasmids was demonstrated using a simple manually operated multipoint inoculator apparatus (MIP) allowing rapid inoculation and later dilution and plating of 25 mating mixtures simultaneously. Forty-five R plasmids belonging to groups F, I, N, and others originally recovered in Escherichia coli K-12 were studied in this as well as in other hosts. The semiquantitative MIP conjugation method was more efficient than conventional matings, particularly when performed in two steps employing E. coli K-12 as intermediate host. Both as donor and as recipient, E. coli K-12 was the most “suitable” general host of the set of plamids studied, although with many plasmids the degree of expression of their transfer functions varied with the host. The expression of fertility in parental bacteria as well as factors in the new host not studied appeared to be of greater importance for the conjugational transfer of a plasmid than the host-specified restriction of plasmid deoxyribonucleic acid by the recipient strain. The MIP conjugation method was successfully used also during screening for transferable R plasmids in gram-negative bacteria present in urine and fecal specimens of humans. The use of a restrictionless mutant instead of a restricting K-12 recipient enabled the detection of additional plasmids. The labor and media-saving MIP conjugation method thus also offers efficiency and is very practical for the performance of large numbers of plasmid matings, for example, in studies of compatibilty, host range, and mobilization of plasmids, as well as for screening purposes.     

Rule-based modelling of conjugative plasmid transfer and incompatibility

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.     

When phage,plasmids, and transposons collide: genomic islands,and conjugative- and mobilizable-transposons as a mosaic continuum

Plasmids and bacteriophage represent the classical vectors for gene transfer within the horizontal gene pool. However, the more recent discovery of an increasing array of other mobile genetic elements (MGE) including genomic islands (GIs), conjugative transposons (CTns), and mobilizable transposons (MTns) which each integrate within the chromosome, offer an increasingly diverse assemblage contributing to bacterial adaptation and evolution. Molecular characterisation of these elements has revealed that they are comprised of functional modules derived from phage, plasmids, and transposons, and further that these modules are combined to generate a continuum of mosaic MGE. In particular, they are comprised of any one of three distinct types of recombinase, together with plasmid-derived transfer and mobilisation gene functions. This review highlights both the similarities and distinctions between these integrating transferable elements resulting from combination of the MGE toolbox.     

Evolution of gene transfer in bacteria

The transfer of genetic information by transformation, conjugation and transduction in bacteria occurs frequently in nature. These diverse gene transfer mechanisms in bacteria are the result of evolution and are not linked to reproduction as in eukaryotic organisms. In this review, gene transfer in bacteria will be considered from an evolutionary perspective.     

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.     

Mobilizable narrow host range plasmids as natural suicide vectors enabling horizontal gene transfer among distantly related bacterial species

Klebsiella pneumoniae 287-w carries three small narrow host range (NHR) plasmids (pIGMS31, pIGMS32, and pIGRK), which could be maintained in several closely related species of Gammaproteobacteria, but not in Alphaproteobacteria. The plasmids contain different mobilization systems (MOB), whose activity in Escherichia coli was demonstrated in the presence of the helper transfer system originating from plasmid RK2. The MOBs of pIGMS31 and pIGMS32 are highly conserved in many bacterial plasmids (members of the MOB family), while the predicted MOB of pIGRK has a unique structure, encoding a protein similar to phage-related integrases. The MOBs of pIGMS31 and pIGMS32 enabled the transfer of heterologous replicons from E. coli into both gammaproteobacterial and alphaproteobacterial hosts, which suggests that these NHR plasmids contain broad host range MOB systems. Such plasmids therefore represent efficient carrier molecules, which may act as natural suicide vectors promoting the spread of diverse genetic information (including other types of mobile elements, e.g. resistance transposons) among evolutionarily distinct bacterial species. Thus, mobilizable NHR plasmids may play a much more important role in horizontal gene transfer than previously thought.     

Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868.

Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide. They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB. The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene. Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp. savastanoi. The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868. We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site. The deletion was caused during the second transposition or by later recombination between the two IS868 copies. Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes. Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution.     

Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, k_ti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The k_ti values ranged between 10⁻¹⁰ and 10⁻¹⁵ ml cells⁻¹ h⁻¹, when estimated under various conditions. Moderate liquid agitation had a favorable effect on k_tf but agitation rates higher than 33 s⁻¹ (intergrated shear force) greatly decreased the value of k_ti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h⁻¹. The rate constant k_ti at temperatures of 10–15°C was as low as the detection limit (10⁻¹⁵ ml cells⁻¹ h⁻¹).     

The genus Amycolatopsis: Indigenous plasmids, cloning vectors and gene transfer systems

The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes. Most of the members of the genus Amycolatopsis are known to produce antibiotics. Additionally, members of this genus have been reported to metabolize aromatic compounds as the sole sources of carbon and energy. Development of genetic manipulation in Amycolatopsis has progressed slowly due to paucity of genetic tools and methods. The occurrence of indigenous plasmids in different species of Amycolatopsis is not very common. Till date, only three indigenous plasmids viz., pMEA100, pMEA300 and pA387 have been reported in Amycolatopsis species. Various vectors based on the indigenous plasmids, pMEA100, pMEA300 and pA387, have been constructed. These vectors have proved useful for molecular genetics studies of actinomycetes. Molecular genetic work with Amycolatopsis strains is not easy, since transformation methods have to be developed, or at least optimized, for each particular strain. Nonetheless, methods for efficient transformation (polyethyleneglycol (PEG) induced protoplast transformation, transformation by electroporation and direct transformation) have been developed and used successfully for the introduction of DNA into several Amycolatopsis species. The construction of plasmid cloning vectors and the development of gene transfer systems has opened up possibilities for studying the molecular genetics of these bacteria.     

Characterization of IS26‐composite transposons and multidrug resistance in conjugative plasmids from Enterobacter cloacae

SHV‐12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, bla_SHV‐12 has rarely been mobilized. Six multidrug‐resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of bla_SHV‐12‐containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β‐lactamase genes (bla_TEM‐1, bla_SHV‐12, bla_CTX‐M‐3 and/or bla_CTX‐M‐14) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26‐bla_SHV‐12‐IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')‐IIc cassette truncated by IS26 was identified in one isolate. Thus, bla_SHV‐12 was obtained from different plasmids through IS26‐mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug‐resistant E. cloacae isolates.     

Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions

In vivo expression technology (IVET) is a genetic strategy for isolating genes expressed in vivo. In order to fully exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusions, which must be recovered from the chromosome of host bacteria. In bacteria for which transductional methods are not available, the recovery of integrated fusion plasmids is problematic and currently limits broad application of IVET. We describe a rapid, single-step, triparental conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium. This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically relevant microbes and may be employed to recover non-replicating and integrated plasmids in other systems. Received: 28 March 1997 / Accepted: 23 May 1997     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.