The impact of genetic elements (gene mobilizers, such as plasmids and transposons) which provide genetic plasticity to bacterial populations in soil, is still poorly understood, and hence this project aimed to assess the occurrence and role of such elements in soil-related systems using exogenous isolations and molecular approaches. The short-term objective was to identify key elements which play a role in conferring gene mobilizing capacity upon natural Gram-negative and Gram-positive bacterial populations. As a long-term objective, we intended to determine whether the elements which play a role in the different systems under study (bulk and wheat or sugarbeet rhizosphere soil, polluted soil, manured soil, the sugarbeet phytosphere) bear similarity to one another, as well as whether stresses imposed on soil, such as heavy metal or manure pressures, enhance the gene mobilizing capacity. To assess this, an easily selectable mobilizable plasmid released into the different systems, was selected. To achieve the objectives, a wide variety of existing or novel methods, e.g. direct endogenous as well as bi- and tri-parental exogenous plasmid isolation methods, PCR screening and analysis of plasmids and total community soil extracts, analysis of plasmid isolates and PCR generation of plasmid-specific probes based on replication- and/or transfer-related sequences (such as the origin of vegetative replication, oriV) were applied. Another long-term objective was to start assessing whether and under which conditions the genetic elements identified actually drive the genetic plasticity in bacterial populations in soil.
A high number of diverse gene mobilizing elements was obtained from each of the different ecosystems studied by the 7 participating groups. Both the direct exogenous isolation method using mercury or antibiotic resistances as selectable markers and the triparental exogenous isolation method were shown to work for Gram-negative bacteria, and to provide a variety of new plasmids for these hosts. The plasmid isolates were found to depend on the isolation methods applied. For instance, very different transferable plasmids were obtained from a manure sample when using biparental exogenous versus endogenous isolation methods. Plasmid identification and characterization methods, including replicon typing using available rep/inc probes, PCR assisted assessment of replicon type, restriction typing and probing as well as host range of self-transfer and mobilization, have allowed for a classification of the plasmids obtained in separate groups. Many of the plasmids with mobilizing capacity are apparently novel, and several (obtained from different soil-related ecosystems) seem to form a hitherto unknown broad-host range group.
A dot blot hybridisation comparison of key plasmid isolates obtained from the different soil-related systems was performed in a direct collaboration between all laboratories, and similarities as well as differences between these isolates have been identified using cluster analysis.
Several specific probes as well as PCR primer systems for the new plasmids were developed using cloning and sequencing of plasmid-specific regions such as the minimum replication region, oriV.
Furthermore, both specific probes and sets of PCR primers have been developed and tested for the detection of plasmids of the Gram-negative broad host range IncN, IncQ, IncP and IncW incompatibility groups. Whereas all IncQ plasmids tested were detectable using such approaches, remarkable diversity was found in plasmids belonging to the other incompatibility groups. In particular, the plasmids hitherto classified as IncW, may belong to at least two sub-groups based on our molecular analysis.
In manure and soil DNA extracts, mobilizing plasmids of a variety of Inc groups have been identified with this approach. Thus, such plasmids probably have a role in mobilization in manure slurries and soils.
A genetically marked organism (GMO), Pseudomonas fluorescens SBW25, was shown to acquire mercury resistance plasmids when present in the sugarbeet phytosphere in the field without the occurrence of any apparent selective pressure. These could potentially enhance the gene mobilizing capacity of the field- released GMO population. The acquisition of plasmids only became detectable at a certain stage of plant development, i.e. about 80-120 days following the release of GMO-coated seeds. This also corresponded to the first time after planting that plasmids could be isolated from the sugarbeet rhizosphere by exogenous methods into Gramnegative hosts. Moreover, host fitness was differentially affected, in a plant growth dependent fashion, by carriage of an indigenous plasmid. These observations point to temporally dependent transfer activity in the rhizosphere.
A mating system consisting of easily selectable donor and recipient bacteria and a self-transmissible and a mobilizable plasmid was used to asses gene mobilization in the different ecosystems under investigation, during the growing season of 1995/1996. Data were obtained showing that transfer (mobilization) occurred preferentially in the rhizospheres of sugarbeet and wheat, as well as in manured soils. In particular soil with recent input of manure was shown to be conducive to transfers.
MAJOR SCIENTIFIC BREAKTHROUGHS:
A variety of diverse plasmids with gene mobilizing capacity amongst mainly Gram-negative hosts has been obtained from the different soil-related environments under study. Several of these mobilizer plasmids did not classify into known incompatibility groups and so have been suggested to belong to hitherto unknown groups. Thus, the soil and related systems harbour a wealth of known as well as novel gene mobilizing elements, the significance of which we are only beginning to understand.
Amongst plasmids of known groups, the finding of plasmids belonging to the IncP-alfa and IncP-beta groups in various stressed and unstressed soils was important, since it corroborated the view that these plasmid groups are important as broad-host range carriers of accessory genes in the bacterial populations under study. A comprehensive molecular characterization of these plasmids showed a high degree of similarity between the IncP-alfa types, whereas the IncP-beta types were more diverse. Furthermore, there was an expected input of mobilizing or mobilizable plasmids of several other known Inc groups (e.g. IncQ) with manure added to soil, which supports the view that manure input can increase the gene mobilizing capacity of soil bacterial populations.
The novel broad host range (BHR) mobilizing plasmids very likely form the core of at least one and likely more separate groups of BHR plasmids, which broadens our current view of BHR plasmid diversity.
Insights have been gained into the previously unknown diversity in plasmids classed to IncP, IncW and IncN groups. The molecular tools developed for this (specific probes and primer systems) have proven to be very useful. In this research area, collaborative work between partners was most rewarding for IncP plasmids. The application of gene probes made from different amplified regions enabled the comparison of a wide variety of IncP plasmids isolated during this study from soils, the rhizosphere and manure across Europe. The data showed a great deal of similarity between isolates, indicating such molecular microbial ecology approaches can be applied broadly. The prevalence of these plasmids in samples obtained from the soil-related ecosystems was then also investigated.
Molecular probes and primers for the specific detection of mobilizing elements directly in soil were obtained following cloning and sequencing of either random plasmid-specific regions or the origins of vegetative replication of key plasmid isolates. The application of one system directly to wheat rhizosphere soil using a PCR approach revealed that prevalence of the specific plasmid was low, i.e. estimated to be below 10 copies per g soil.
A genetically marked bacterium released into the field (sugarbeet phytosphere) was shown to naturally acquire mercury resistance plasmids in the absence of any known selective pressure for this trait. Thus, plasmid transfer for phenotype can occur in the total absence of selection pressure for some or all of the plasmid phenotypes. This conclusion was confirmed in field studies showing that the mobilization of small plasmids by natural soil replicons could occur without selective pressure at significant frequencies (1E-4-1E-5 transconjugants per recipient). Evidence was also found, at two sites, for preferential plasmid acquisition in late summer, when sugarbeet plants were older. A second release with the same strain studied the impact of carriage of an indigenous plasmid on host ecology. Host fitness was shown to be affected in a plant growth dependent way.
It is likely that gene transfer to/from modified bacteria will eventually take place in the soil environment due to the presence of gene mobilizers. Transfer systems to study such events have been based on non-engineered strains and (newly isolated) selectable plasmids, which allowed for release studies in soil in the growing seasons of 1995 and 1996. Transfer of the mobilizable element was found in manured soil, the wheat rhizosphere as well as the sugarbeet phytosphere.
Funding SchemeCSC - Cost-sharing contracts
OX1 3SR Oxford
CF1 3TL Cardiff