Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments

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 M CdCl 2 ), permitting long-term community monitoring. The broad-host-range IncP␣ 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 Cd r or Ni r 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 Ni r 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.

mentioning

confidence: 84%

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

Smets

Morrow²,

Pinedo³

2003

“…4), which was not fully developed until 6 days after the onset of germination, i.e., on the same day that the first transconjugants were detectable. Some studies indicate that plasmid transfer happens most efficiently when the availability of bacterial substrate is relatively high (29), and for the TOL plasmid it has been demonstrated that the physiological state of the recipient cells influence their ability to participate in transfer (7). We suggest that high concentrations of plant exudates around the root and the hypocotyl causes a high density of donors and putative recipients and perhaps also enhanced plasmid transfer and that these factors together explain why the large majority of transconjugants was observed in these zones of the seedlings (Fig.…”

Section: Discussionmentioning

confidence: 99%

Plasmid Transfer from Pseudomonas putida to the Indigenous Bacteria on Alfalfa Sprouts: Characterization, Direct Quantification, and In Situ Location of Transconjugant Cells

Mølbak

Licht

Kvist

et al. 2003

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 ؋ 10 ؊4 and 2.0 ؋ 10 ؊6 transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and Erwinia. The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.

“…Microbial communities of the deep subsurface are constrained by the scarcity of growth substrates (60), water availability, and spatial separation (77), which result in low metabolic rates (28) and the dominance of chemolithoautotrophic metabolism (10,77). Because population densities (52) and metabolic activities (72) stimulate LGT, this process may be limited in the deep subsurface. Nevertheless, anecdotal evidence indicates that LGT affects the genetic and metabolic diversities of subsurface microbial communities.…”

mentioning

confidence: 99%

Molecular Evidence for the Evolution of Metal Homeostasis Genes by Lateral Gene Transfer in Bacteria from the Deep Terrestrial Subsurface

Coombs

Barkay

2004

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P IB -type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P IB -type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the ␤ subclass of the Proteobacteria (␤-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of ␤-and ␥-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G؉C content of the P IB -type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.The role of lateral gene transfer (LGT) in the evolution of microorganisms becomes more and more apparent with every new microbial genome that is sequenced and annotated (54, 58), which has led many workers to question our basic concepts of microbial speciation (18, 23). The prevalence of laterally transferred genes clearly suggests that this mode of evolution is advantageous to microbial life, possibly by providing the means for genetic innovation in the absence of frequent sexual recombination (37). This variation is likely to increase metabolic diversity, and consequently competence, in environments subject to frequent change (13, 61). The possibility that LGT is an important force in shaping the structure and function of microbial communities in their natural habitats is suggested by (i) the fact that abundant and diverse plasmids (12, 73), viruses (8, 89), insertion sequences (70), transposons (59, 78), integrons (49), and other elements that contribute to genomic plasticity are commonly isolated from environmental samples and strains; (ii) the fact that natural competence is common among microbes (11) and occurs in soils (17, 50) and natural water (88); (iii) the fact that LGT has been demonstrated in model ecosystems (i.e., microcosms) (52, 62, 75, 80) and in intact environmental samples (19); and (iv) the fact that seeding soils with bacteria carrying conjugal catabolic plasmids results in transfer of the plasmids to indigenous soil microbes and the stimulation of plasmid-specified activit...