Ralstonia solanacearum virulence increased following large interstrain gene transfers by natural transformation.

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.

Antibiotic-resistant soil bacteria in transgenic plant fields.

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

Detection of potential transgenic plant DNA recipients among soil bacteria.

The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.