Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Phytoplanktonic species in the haloalkaline Lake Dziani Dzaha select their archaeal microbiome.

Microorganisms are key contributors of aquatic biogeochemical cycles but their microscale ecology remains largely unexplored, especially interactions occurring between phytoplankton and microorganisms in the phycosphere, that is the region immediately surrounding phytoplankton cells. The current study aimed to provide evidence of the phycosphere taking advantage of a unique hypersaline, hyperalkaline ecosystem, Lake Dziani Dzaha (Mayotte), where two phytoplanktonic species permanently co-dominate: a cyanobacterium, Arthrospira fusiformis, and a green microalga, Picocystis salinarum. To assay phycospheric microbial diversity from in situ sampling, we set up a flow cytometry cell-sorting methodology for both phytoplanktonic populations, coupled with metabarcoding and comparative microbiome diversity. We focused on archaeal communities as they represent a non-negligible part of the phycospheric diversity, however their role is poorly understood. This work is the first which successfully explores in situ archaeal diversity distribution showing contrasted phycospheric compositions, with P. salinarum phycosphere notably enriched in Woesearchaeales OTUs while A. fusiformis phycosphere was enriched in methanogenic lineages affiliated OTUs such as Methanomicrobiales or Methanofastidiosales. Most archaeal OTUs, including Woesearchaeales considered in literature as symbionts, were either ubiquitous or specific of the free-living microbiome (i.e. present in the 3-0.2 µm fraction). Seminally, several archaeal OTUs were enriched from the free-living microbiome to the phytoplankton phycospheres, suggesting (i) either the inhibition or decrease of other OTUs, or (ii) the selection of specific OTUs resulting from the physical influence of phytoplanktonic species on surrounding Archaea.

Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

A deep-sea bacterium related to coastal marine pathogens.

Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here, we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309 m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptation to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infections in farmed oysters. The results of functional in vitro assays on immunocytes (haemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, which might have played an evolutionary role in the origin of coastal marine pathogens.

Vibrio splendidus O-antigen structure: a trade-off between virulence to oysters and resistance to grazers.

A major debate in evolutionary biology is whether virulence is maintained as an adaptive trait and/or evolves to non-virulence. In the environment, virulence traits of non-obligatory parasites are subjected to diverse selective pressures and trade-offs. Here, we focus on a population of Vibrio splendidus that displays moderate virulence for oysters. A MARTX (Multifunctional-autoprocessing repeats-in-toxin) and a type-six secretion system (T6SS) were found to be necessary for virulence toward oysters, while a region (wbe) involved in O-antigen synthesis is necessary for resistance to predation against amoebae. Gene inactivation within the wbe region had major consequences on the O-antigen structure, conferring lower immunogenicity, competitive advantage and increased virulence in oyster experimental infections. Therefore, O-antigen structures that favour resistance to environmental predators result in an increased activation of the oyster immune system and a reduced virulence in that host. These trade-offs likely contribute to maintaining O-antigen diversity in the marine environment by favouring genomic plasticity of the wbe region. The results of this study indicate an evolution of V. splendidus towards moderate virulence as a compromise between fitness in the oyster as a host, and resistance to its predators in the environment.

Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells.

Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the sustainability of commercial and natural stocks of this species. Vibrio crassostreae (V. crassostreae) has been repeatedly isolated from diseased animals, and the majority of the strains have been demonstrated to be virulent for oysters. In this study, we showed that oyster farms exhibited a high prevalence of a virulence plasmid carried by V. crassostreae, while oysters, at an adult stage, were reservoirs of this virulent population. The pathogenicity of V. crassostreae depends on a novel transcriptional regulator, which activates the bidirectional promoter of a type 6 secretion system (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are necessary for virulence but act independently to cause haemocyte (oyster immune cell) cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. tapetis, which infect adult oysters and clams respectively. We propose that haemocyte cytotoxicity is a lethality trait shared by a broad range of mollusc pathogens, and we speculate that T6SS was involved in parallel evolution of pathogen for molluscs.

Contrasted ecological niches shape fungal and prokaryotic community structure in mangroves sediments.

Mangroves are forest ecosystems located at the interface between land and sea where sediments presented a variety of contrasted environmental conditions (i.e. oxic/anoxic, non-sulfidic/sulfidic, organic matter content) providing an ideal ecosystem to study microbial communities with niche differentiation and distinct community structures. In this work, prokaryotic and fungal compositions were investigated during both wet and dry seasons in New Caledonian mangrove sediments, from the surface to deeper horizons under the two most common tree species in this region (Avicennia marina and Rhizophora stylosa), using high-throughput sequencing. Our results showed that Bacteria and Archaea communities were mainly shaped by sediment depth while the fungal community was almost evenly distributed according to sediment depth, vegetation cover and season. A detailed analysis of prokaryotic and fungal phyla showed a dominance of Ascomycota over Basidiomycota whatever the compartment, while there was a clear shift in prokaryotic composition. Some prokaryotic phyla were enriched in surface layers such as Proteobacteria, Euryarchaeota while others were mostly associated with deeper layers as Chloroflexi, Bathyarchaeota, Aminicenantes. Our results highlight the importance of considering fungal and prokaryotic counterparts for a better understanding of the microbial succession involved in plant organic matter decomposition in tropical coastal sediments.

Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes.

Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes-as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.

Transcriptomic insights into the dominance of two phototrophs throughout the water column of a tropical hypersaline-alkaline crater lake (Dziani Dzaha, Mayotte).

Saline-alkaline lakes often shelter high biomasses despite challenging conditions, owing to the occurrence of highly adapted phototrophs. Dziani Dzaha (Mayotte) is one such lake characterized by the stable co-dominance of the cyanobacterium Limnospira platensis and the picoeukaryote Picocystis salinarum throughout its water column. Despite light penetrating only into the uppermost meter, the prevailing co-dominance of these species persists even in light- and oxygen-deprived zones. Here, a depth profile of phototrophs metatranscriptomes, annotated using genomic data from isolated strains, is employed to identify expression patterns of genes related to carbon processing pathways including photosynthesis, transporters and fermentation. The findings indicate a prominence of gene expression associated with photosynthesis, with a peak of expression around 1 m below the surface, although the light intensity is very low and only red and dark red wavelengths can reach it, given the very high turbidity linked to the high biomass of L. platensis. Experiments on strains confirmed that both species do grow under these wavelengths, at rates comparable to those obtained under white light. A decrease in the expression of photosynthesis-related genes was observed in L. platensis with increasing depth, whereas P. salinarum maintained a very high pool of psbA transcripts down to the deepest point as a possible adaptation against photodamage, in the absence and/or very low levels of expression of genes involved in protection. In the aphotic/anoxic zone, expression of genes involved in fermentation pathways suggests active metabolism of reserve or available dissolved carbon compounds. Overall, L. platensis seems to be adapted to the uppermost water layer, where it is probably maintained thanks to gas vesicles, as evidenced by high expression of the gvpA gene. In contrast, P. salinarum occurs at similar densities throughout the water column, with a peak in abundance and gene expression levels which suggests a better adaptation to lower light intensities. These slight differences may contribute to limited inter-specific competition, favoring stable co-dominance of these two phototrophs.

Unexpected phytostimulatory behavior for Escherichia coli and Agrobacterium tumefaciens model strains.

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

Metabarcoding of the Three Domains of Life in Aquatic Saline Ecosystems.

High-throughput amplicon sequencing, known as metabarcoding, is a powerful technique to decipher exhaustive microbial diversity considering specific gene markers. While most of the studies investigating ecosystem functioning through microbial diversity targeted only one domain of life, either bacteria, or archaea or microeukaryotes, the remaining challenge in microbial ecology is to uncover the integrated view of microbial diversity occurring in ecosystems. Indeed, interactions occurring between the different microbial counterparts are now recognized having a great impact on stability and resilience of ecosystems. Here, we summarize protocols describing sampling, molecular, and simultaneous metabarcoding of bacteria, archaea, and microeukaryotes, as well as a bioinformatic pipeline allowing the study of exhaustive microbial diversity in natural aquatic saline samples.

Strong reorganization of multi-domain microbial networks associated with primary producers sedimentation from oxic to anoxic conditions in an hypersaline lake.

Understanding the role of microbial interactions in the functioning of natural systems is often impaired by the levels of complexity they encompass. In this study, we used the relative simplicity of an hypersaline crater lake hosting only microbial organisms (Dziani Dzaha) to provide a detailed analysis of the microbial networks including the three domains of life. We identified two main ecological zones, one euphotic and oxic zone in surface, where two phytoplanktonic organisms produce a very high biomass, and one aphotic and anoxic deeper zone, where this biomass slowly sinks and undergoes anaerobic degradation. We highlighted strong differences in the structure of microbial communities from the two zones and between the microbial consortia associated with the two primary producers. Primary producers sedimentation was associated with a major reorganization of the microbial network at several levels: global properties, modules composition, nodes and links characteristics. We evidenced the potential dependency of Woesearchaeota to the primary producers' exudates in the surface zone, and their disappearance in the deeper anoxic zone, along with the restructuration of the networks in the anoxic zone toward the decomposition of the organic matter. Altogether, we provided an in-depth analysis of microbial association network and highlighted putative changes in microbial interactions supporting the functioning of the two ecological zones in this unique ecosystem.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Effects of the Denitrification Inhibitor "Procyanidins" on the Diversity, Interactions, and Potential Functions of Rhizosphere-Associated Microbiome.

Some plant secondary metabolites, such as procyanidins, have been demonstrated to cause biological denitrification inhibition (BDI) of denitrifiers in soils concomitantly with a gain in plant biomass. The present work evaluated whether procyanidins had an impact on the diversity of nontarget microbial communities that are probably involved in soil fertility and ecosystem services. Lettuce plants were grown in two contrasting soils, namely Manziat (a loamy sand soil) and Serail (a sandy clay loam soil) with and without procyanidin amendment. Microbial diversity was assessed using Illumina sequencing of prokaryotic 16S rRNA gene and fungal ITS regions. We used a functional inference to evaluate the putative microbial functions present in both soils and reconstructed the microbial interaction network. The results showed a segregation of soil microbiomes present in Serail and Manziat that were dependent on specific soil edaphic variables. For example, Deltaproteobacteria was related to total nitrogen content in Manziat, while Leotiomycetes and Firmicutes were linked to Ca2+ in Serail. Procyanidin amendment did not affect the diversity and putative activity of microbial communities. In contrast, microbial interactions differed according to procyanidin amendment, with the results showing an enrichment of Entotheonellaeota and Mucoromycota in Serail soil and of Dependentiae and Rozellomycetes in Manziat soil.

Molecular Targets for Coevolutionary Interactions Between Pacific Oyster Larvae and Their Sympatric Vibrios.

Bacteria of the Vibrio genus are the most predominant infectious agents threatening marine wildlife and aquaculture. Due to the large genetic diversity of these pathogens, the molecular determinants of Vibrio virulence are only poorly understood. Furthermore, studies tend to ignore co-evolutionary interactions between different host populations and their locally encountered Vibrio communities. Here, we explore the molecular targets of such co-evolutionary interactions by analyzing the genomes of nine Vibrio strains from the Splendidus-clade showing opposite virulence patterns towards two populations of Pacific oysters introduced into European Wadden Sea. By contrasting Vibrio phylogeny to their host specific virulence patterns, we could identify two core genome genes (OG1907 and OG 3159) that determine the genotype by genotype (G × G) interactions between oyster larvae and their sympatric Vibrio communities. Both genes show positive selection between locations targeting only few amino acid positions. Deletion of each gene led to a loss of the host specific virulence patterns while complementation with OG3159 alleles from both locations could recreate the wild type phenotypes matching the origin of the allele. This indicates that both genes can act as a genetic switch for Vibrio-oyster coevolution demonstrating that local adaptation in distinct Vibrio lineages can rely on only few genes independent of larger pathogenicity islands or plasmids.

Ancestral gene acquisition as the key to virulence potential in environmental Vibrio populations.

Diseases of marine animals caused by bacteria of the genus Vibrio are on the rise worldwide. Understanding the eco-evolutionary dynamics of these infectious agents is important for predicting and managing these diseases. Yet, compared to Vibrio infecting humans, knowledge of their role as animal pathogens is scarce. Here we ask how widespread is virulence among ecologically differentiated Vibrio populations, and what is the nature and frequency of virulence genes within these populations? We use a combination of population genomics and molecular genetics to assay hundreds of Vibrio strains for their virulence in the oyster Crassostrea gigas, a unique animal model that allows high-throughput infection assays. We show that within the diverse Splendidus clade, virulence represents an ancestral trait but has been lost from several populations. Two loci are necessary for virulence, the first being widely distributed across the Splendidus clade and consisting of an exported conserved protein (R5.7). The second is a MARTX toxin cluster, which only occurs within V. splendidus and is for the first time associated with virulence in marine invertebrates. Varying frequencies of both loci among populations indicate different selective pressures and alternative ecological roles, based on which we suggest strategies for epidemiological surveys.

Distribution of 2,4-Diacetylphloroglucinol Biosynthetic Genes among the Pseudomonas spp. Reveals Unexpected Polyphyletism.

Fluorescent pseudomonads protecting plant roots from phytopathogens by producing 2,4-diacetylphloroglucinol (DAPG) are considered to form a monophyletic lineage comprised of DAPG+Pseudomonas strains in the "P. corrugata" and "P. protegens" subgroups of the "Pseudomonas fluorescens" group. However, DAPG production ability has not been investigated for many species of these two subgroups, and whether or not the DAPG+Pseudomonas are truly monophyletic remained to be verified. Thus, the distribution of the DAPG biosynthetic operon (phlACBD genes) in the Pseudomonas spp. was investigated in sequenced genomes and type strains. Results showed that the DAPG+Pseudomonas include species of the "P. fluorescens" group, i.e., P. protegens, P. brassicacearum, P. kilonensis, and P. thivervalensis, as expected, as well as P. gingeri in which it had not been documented. Surprisingly, they also include bacteria outside the "P. fluorescens" group, as exemplified by Pseudomonas sp. OT69, and even two Betaproteobacteria genera. The phl operon-based phylogenetic tree was substantially congruent with the one inferred from concatenated housekeeping genes rpoB, gyrB, and rrs. Contrariwise to current supposition, ancestral character reconstructions favored multiple independent acquisitions rather that one ancestral event followed by vertical inheritance. Indeed, based on synteny analyses, these acquisitions appeared to vary according to the Pseudomonas subgroup and even the phylogenetic groups within the subgroups. In conclusion, our study shows that the phl+Pseudomonas populations form a polyphyletic group and suggests that DAPG biosynthesis might not be restricted to this genus. This is important to consider when assessing the ecological significance of phl+ bacterial populations in rhizosphere ecosystems.

Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition.

Vibrios are frequently associated with oyster mortality; however whether they are the primary causative agent or secondary opportunistic colonizers is not well understood. Here we combine analysis of natural infection dynamics, population genomics and molecular genetics to ask (i) to what extent oysters are passively colonized by Vibrio population present in the surrounding water, (ii) how populations turn over during pathogenicity events and (iii) what genetic factors are responsible for pathogenicity. We identified several populations of Vibrio preferentially associated with oyster tissues. Among these, Vibrio crassostreae is particularly abundant in diseased animals while nearly absent in the surrounding water, and its pathogenicity is correlated with the presence of a large mobilizable plasmid. We further demonstrate that the plasmid is essential for killing but not necessary for survival in tissues of oysters. Our results suggest that V. crassostreae first differentiated into a benign oyster colonizer that was secondarily turned into a pathogen by introgression of a virulence plasmid into the population, possibly facilitated by elevated host density in farming areas.

Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease.

Successive disease outbreaks in oyster (Crassostrea gigas) beds in France have resulted in dramatic losses in production, and subsequent decline in the oyster-farming industry. Deaths of juvenile oysters have been associated with the presence of a herpes virus (OsHV-1 µvar) and bacterial populations of the genus Vibrio. Although the pathogenicity of OsHV-1 µvar, as well as several strains of Vibrio has been demonstrated by experimental infections, our understanding of the complexity of infections occurring in the natural environment remains limited. In the present study, we use specific-pathogen-free (SPF) oysters infected in an estuarine environment to study the diversity and dynamics of cultured microbial populations during disease expression. We observe that rapid Vibrio colonization followed by viral replication precedes oyster death. No correlation was found between the vibrio concentration and viral load in co-infected animals. We show that the quantity of viral DNA is a predictor of mortality, however, in the absence of bacteria, a high load of herpes virus is not sufficient to induce the full expression of the disease. In addition, we demonstrate that juvenile mortalities can occur in the absence of herpes virus, indicating that the herpes virus appears neither essential nor sufficient to cause juvenile deaths; whereas bacteria are necessary for the disease. Finally, we demonstrate that oysters are a reservoir of putative pathogens, and that the geographic origin, age, and cultivation method of oysters influence disease expression.

Analysis of genes contributing to plant-beneficial functions in Plant Growth-Promoting Rhizobacteria and related Proteobacteria.

The positive effects of root-colonizing bacteria cooperating with plants lead to improved growth and/or health of their eukaryotic hosts. Some of these Plant Growth-Promoting Rhizobacteria (PGPR) display several plant-beneficial properties, suggesting that the accumulation of the corresponding genes could have been selected in these bacteria. Here, this issue was targeted using 23 genes contributing directly or indirectly to established PGPR effects, based on genome sequence analysis of 304 contrasted Alpha- Beta- and Gammaproteobacteria. Most of the 23 genes studied were also found in non-PGPR Proteobacteria and none of them were common to all 25 PGPR genomes studied. However, ancestral character reconstruction indicated that gene transfers -predominantly ancient- resulted in characteristic gene combinations according to taxonomic subgroups of PGPR strains. This suggests that the PGPR-plant cooperation could have established separately in various taxa, yielding PGPR strains that use different gene assortments. The number of genes contributing to plant-beneficial functions increased along the continuum -animal pathogens, phytopathogens, saprophytes, endophytes/symbionts, PGPR- indicating that the accumulation of these genes (and possibly of different plant-beneficial traits) might be an intrinsic PGPR feature. This work uncovered preferential associations occurring between certain genes contributing to phytobeneficial traits and provides new insights into the emergence of PGPR bacteria.