Horizontal Gene Transfer Regulation in Bacteria as a “Spandrel” of DNA Repair Mechanisms

Horizontal gene transfer (HGT) is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3%) of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic “hot spots”, which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination “hot-spots” to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment might have led the bacteria to hijack DNA repair mechanisms in order to generate genetic diversity without losing too much genomic stability

Homologous Recombination in Agrobacterium: Potential Implications for the Genomic Species Concept in Bacteria

International audienceAccording to current taxonomical rules, a bona fide bacterial species is a genomic species characterized by the genomic similarity of its members. It has been proposed that the genomic cohesion of such clusters may be related to sexual isolation, which limits gene flow between too divergent bacteria. Homologous recombination is one of the most studied mechanisms responsible for this genetic isolation. Previous studies on several bacterial models showed that recombination frequencies decreased exponentially with increasing DNA sequence divergence. In the present study, we investigated this relationship in the Agrobacterium tumefaciens species complex, which allowed us to focus on sequence divergence in the vicinity of the genetic boundaries of genomic species. We observed that the sensitivity of the recombination frequency to DNA divergence fitted a log-linear function until approximately 10% sequence divergence. The results clearly revealed that there was no sharp drop in recombination frequencies at the point where the sequence divergence distribution showed a ‘‘gap'' delineating genomic species. The ratio of the recombination frequency in homogamic conditions relative to this frequency in heterogamic conditions, that is, sexual isolation, was found to decrease from 8 between the most distant strains within a species to 9 between the most closely related species, for respective increases from 4.3% to 6.4% mismatches in the marker gene chvA. This means that there was only a 1.13-fold decrease in recombination frequencies for recombination events at both edges of the species border. Hence, from the findings of this investigation, we conclude that—at least in this taxon—sexual isolation based on homologous recombination is likely not high enough to strongly hamper gene flow between species as compared with gene flow between distantly related members of the same species. The 70% relative binding ratio cutoff used to define bacterial species is likely correlated to only minor declines in homologous recombination frequencies. Consequently, the sequence diversity, as a mechanistic factor for the efficiency of recombination (as assayed in the laboratory), appears to play little role in the genetic cohesion of bacterial species, and thus, the genomic species definition for prokaryotes is definitively not reconcilable with the biological species concept for eukaryotes

Potential Dissemination of Antibiotic Resistance Genes From Transgenic Plants to Microorganisms

International audienceEvidence that genes were transferred during evolution from plants to bacteria was obtained from nucleotide and protein sequence analyses. However, the extent of such transfers among phylogenetically distant organisms is limited by various factors, including those related to complexity of the environment and those endogenous to the bacteria, designed to prevent a drift of the genome integrity. The goal of this article is to give an overview of the potentials and limits of natural interkingdom gene transfers, with a particular focus on prokaryote originating sequences fitting the nuclear genome of transgenic plants

In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes

International audienceThe essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae. No practical means of disease control or preventionagainst this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal genetransfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians

A Comprehensive Overview of the Genes and Functions Required for Lettuce Infection by the Hemibiotrophic Phytopathogen Xanthomonas hortorum pv. vitians

International audienceThe successful infection of a host plant by a phytopathogenic bacterium depends on a finely tuned molecular cross talk between the two partners. Thanks to transposon insertion sequencing techniques (Tn-seq), whole genomes can now be assessed to determine which genes are important for the fitness of several plant-associated bacteria in planta. Despite its agricultural relevance, the dynamic molecular interaction established between the foliar hemibiotrophic phytopathogen Xanthomonas hortorum pv. vitians and its host, lettuce (Lactuca sativa), remains completely unknown. To decipher the genes and functions mobilized by the pathogen throughout the infection process, we conducted a Tn-seq experiment in lettuce leaves to mimic the selective pressure occurring during natural infection. This genome-wide screening identified 170 genes whose disruption caused serious fitness defects in lettuce. A thorough examination of these genes using comparative genomics and gene set enrichment analyses highlighted that several functions and pathways were highly critical for the pathogen's survival. Numerous genes involved in amino acid, nucleic acid, and exopolysaccharide biosynthesis were critical. The xps type II secretion system operon, a few TonB-dependent transporters involved in carbohydrate or siderophore scavenging, and multiple genes of the carbohydrate catabolism pathways were also critical, emphasizing the importance of nutrition systems in a nutrient-limited environment. Finally, several genes implied in camouflage from the plant immune system and resistance to immunity-induced oxidative stress were strongly involved in host colonization. As a whole, these results highlight some of the central metabolic pathways and cellular functions critical for Xanthomonas host adaptation and pathogenesis.IMPORTANCE Xanthomonas hortorum was recently the subject of renewed interest, as several studies highlighted that its members were responsible for diseases in a wide range of plant species, including crops of agricultural relevance (e.g., tomato and carrot). Among X. hortorum variants, X. hortorum pv. vitians is a reemerging foliar hemibiotrophic phytopathogen responsible for severe outbreaks of bacterial leaf spot of lettuce all around the world. Despite recent findings, sustainable and practical means of disease control remain to be developed. Understanding the host-pathogen interaction from a molecular perspective is crucial to support these efforts. The genes and functions mobilized by X. hortorum pv. vitians during its interaction with lettuce had never been investigated. Our study sheds light on these processes by screening the whole pathogen genome for genes critical for its fitness during the infection process, using transposon insertion sequencing and comparative genomics

Natural transformation-based foreign DNA acquisition in a Ralstonia solanacearum mutS mutant

International audienceMutator strains with defective methyl-mismatch repair (MMR) systems have been shown to play an important role in adaptation of bacterial populations to changing and stressful environments. In this report, we describe the impact of mutS::aacC3-IV inactivation on foreign DNA acquisition by natural transformation in the phytopathogenic bacterium Ralstonia solanacearum. A mutS mutant of R. solanacearum exhibited 33- to 60-fold greater spontaneous mutation frequencies, in accordance with a mutator phenotype. Transformation experiments indicated that intra- and interspecific DNA transfers increased up to 89-fold. To assess horizontal gene transfer (HGT) from genetically modified plants to R. solanacearum, fitness of the mutator was first evaluated in soil and plant environments. Competitiveness was not modified after 61 days in soil and 8 days in tomato, and the progress of plant decay symptoms was similar to that of the wild-type strain. Despite its survival in soil and in planta, and the powerful capacities of HGT, R. solanacearum was not genetically transformed by transgenic plant DNA in a wide range of in vitro and in planta tests. 2007 Elsevier Masson SAS. All rights reserved

Impact of the Microscale Distribution of a Pseudomonas Strain Introduced into Soil on Potential Contacts with Indigenous Bacteria

Soil bioaugmentation is a promising approach in soil bioremediation and agriculture. Nevertheless, our knowledge of the fate and activity of introduced bacteria in soil and thus of their impact on the soil environment is still limited. The microscale spatial distribution of introduced bacteria has rarely been studied, although it determines the encounter probability between introduced cells and any components of the soil ecosystem and thus plays a role in the ecology of introduced bacteria. For example, conjugal gene transfer from introduced bacteria to indigenous bacteria requires cell-to-cell contact, the probability of which depends on their spatial distribution. To quantitatively characterize the microscale distribution of an introduced bacterial population and its dynamics, a gfp-tagged derivative of Pseudomonas putida KT2440 was introduced by percolation in repacked soil columns. Initially, the introduced population was less widely spread at the microscale level than two model indigenous functional communities: the 2,4-dichlorophenoxyacetic acid degraders and the nitrifiers (each at 10(6) CFU g(−1) soil). When the soil was percolated with a substrate metabolizable by P. putida or incubated for 1 month, the microscale distribution of introduced bacteria was modified towards a more widely dispersed distribution. The quantitative data indicate that the microscale spatial distribution of an introduced strain may strongly limit its contacts with the members of an indigenous bacterial community. This could constitute an explanation to the low number of indigenous transconjugants found most of time when a plasmid-donor strain is introduced into soil

Horizontal gene transfer between Ralstonia solanacearum strains detected by comparative genomic hybridization on microarrays

International audienceThe plant pathogenic Betaproteobacterium Ralstonia solanacearum is a complex species in that most of the strains share the common characteristic of being naturally transformable. In this study, we used a new approach based on comparative genomic hybridization (CGH) on microarrays to investigate the extent of horizontal gene transfers (HGTs) between different strains of R. solanacearum. Recipient strains from phylotypes I, II and III were naturally transformed in vitro by genomic DNA from the GMI1000 reference strain (phylotype I) and the resulting DNAs were hybridized on a microarray representative of the 5120 predicted genes from the GMI1000 strain. In addition to transfer of the antibiotic resistance marker, in 8 of the 16 tested transformants, CGH on microarrays detected other transferred GMI1000 genes and revealed their number, category, function and localization along the genome. We showed that DNA blocks up to 30 kb and 33 genes could be integrated during a single event. Most of these blocks flanked the marker gene DNA but, interestingly, multiple DNA acquisitions along the genome also occurred in a single recombinant clone in one transformation experiment. The results were confirmed by PCR amplification, cloning and sequencing and Southern blot hybridization. This represents the first comprehensive identification of gene acquisitions and losses along the genome of the recipient bacterial strain during natural transformation experiments. In future studies, this strategy should help to answer many questions related to HGT mechanisms