Francesco Pini

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti

BackgroundSinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains.ResultsWith sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains.ConclusionsIn conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.

Replicon-dependent bacterial genome evolution: the case of Sinorhizobium meliloti.

Many bacterial species, such as the alphaproteobacterium Sinorhizobium meliloti, are characterized by open pangenomes and contain multipartite genomes consisting of a chromosome and other large-sized replicons, such as chromids, megaplasmids, and plasmids. The evolutionary forces in both functional and structural aspects that shape the pangenome of species with multipartite genomes are still poorly understood. Therefore, we sequenced the genomes of 10 new S. meliloti strains, analyzed with four publicly available additional genomic sequences. Results indicated that the three main replicons present in these strains (a chromosome, a chromid, and a megaplasmid) partly show replicon-specific behaviors related to strain differentiation. In particular, the pSymB chromid was shown to be a hot spot for positively selected genes, and, unexpectedly, genes resident in the pSymB chromid were also found to be more widespread in distant taxa than those located in the other replicons. Moreover, through the exploitation of a DNA proximity network, a series of conserved “DNA backbones” were found to shape the evolution of the genome structure, with the rest of the genome experiencing rearrangements. The presented data allow depicting a scenario where the pSymB chromid has a distinctive role in intraspecies differentiation and in evolution through positive selection, whereas the pSymA megaplasmid mostly contributes to structural fluidity and to the emergence of new functions, indicating a specific evolutionary role for each replicon in the pangenome evolution.

Exploring the plant-associated bacterial communities in Medicago sativa L

BackgroundPlant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti) level.ResultsResults, obtained by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis, quantitative PCR and sequencing of 16 S rRNA gene libraries, showed a high taxonomic diversity as well as a dominance by members of the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant tissues, while soil Alphaproteobacteria were represented by the families of Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At the single species level, we were able to detect the presence of S. meliloti populations in aerial tissues, nodules and soil. An analysis of population diversity on nodules and soil showed a relatively low sharing of haplotypes (30-40%) between the two environments and between replicate mesocosms, suggesting drift as main force shaping S. meliloti population at least in this system.ConclusionsIn this work we shed some light on the bacterial communities associated with M. sativa plants, showing that Alphaproteobacteria may constitute an important part of biodiversity in this system, which includes also the well known symbiont S. meliloti. Interestingly, this last species was also found in plant aerial part, by applying cultivation-independent protocols, and a genetic diversity analysis suggested that population structure could be strongly influenced by random drift.

Plant-Bacteria Association and Symbiosis: Are There Common Genomic Traits in Alphaproteobacteria?

Alphaproteobacteria show a great versatility in adapting to a broad range of environments and lifestyles, with the association between bacteria and plants as one of the most intriguing, spanning from relatively unspecific nonsymbiotic association (as rhizospheric or endophytic strains) to the highly species-specific interaction of rhizobia. To shed some light on possible common genetic features in such a heterogeneous set of plant associations, the genomes of 92 Alphaproteobacteria strains were analyzed with a fuzzy orthologs-species detection approach. This showed that the different habitats and lifestyles of plant-associated bacteria (soil, plant colonizers, symbiont) are partially reflected by the trend to have larger genomes with respect to nonplant-associated species. A relatively large set of genes specific to symbiotic bacteria (73 orthologous groups) was found, with a remarkable presence of regulators, sugar transporters, metabolic enzymes, nodulation genes and several genes with unknown function that could be good candidates for further characterization. Interestingly, 15 orthologous groupspresent in all plant-associated bacteria (symbiotic and nonsymbiotic), but absent in nonplant-associated bacteria, were also found, whose functions were mainly related to regulation of gene expression and electron transport. Two of these orthologous groups were also detected in fully sequenced plant-associated Betaproteobacteria and Gammaproteobacteria. Overall these results lead us to hypothesize that plant-bacteria associations, though quite variable, are partially supported by a conserved set of unsuspected gene functions.

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.

Plant-by-Plant Variations of Bacterial Communities Associated with Leaves of the Nickel Hyperaccumulator Alyssum bertolonii Desv.

Bacteria associated with tissues of metal-hyperaccumulating plants are of great interest due to the multiple roles they may play with respect to plant growth and resistance to heavy metals. The variability of bacterial communities associated with plant tissues of three populations of Alyssum bertolonii, a Ni hyperaccumulator endemic of serpentine outcrops of Central Italy, was investigated. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes was applied to DNA extracted from leaf tissues of 30 individual plants from three geographically separated serpentine outcrops. Moreover, T-RFLP fingerprinting was also performed on DNA extracted from the same soils from which the plants were collected. Fifty-nine unique terminal-restriction fragments (TRFs) were identified, with more than half of the taxonomically interpreted TRFs assigned to Alpha- and Gamma-Proteobacteria and Clostridia. Data were then used to define the extent of variation of bacterial communities due to single plants or to plant populations. Results indicated a very high plant-by-plant variation of leaf-associated community (more than 93% of total variance observed). However, a core (numerically small) of plant-specific TRFs was found. This work demonstrates that plant-associated bacterial communities represent a large reservoir of biodiversity and that the high variability existing between plants, even from the same population, should be taken into account in future studies on association between bacteria and metal-hyperaccumulating plants.

Development of real-time PCR assay for detection and quantification of Sinorhizobium meliloti in soil and plant tissue.

Aims:  Sinorhizobium meliloti is a nitrogen‐fixing alpha‐proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most‐probable‐number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real‐time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species‐specific fashion, the chromosomal gene rpoE1 and the pSymA gene nodC.

Evolution of Intra-specific Regulatory Networks in a Multipartite Bacterial Genome

Reconstruction of the regulatory network is an important step in understanding how organisms control the expression of gene products and therefore phenotypes. Recent studies have pointed out the importance of regulatory network plasticity in bacterial adaptation and evolution. The evolution of such networks within and outside the species boundary is however still obscure. Sinorhizobium meliloti is an ideal species for such study, having three large replicons, many genomes available and a significant knowledge of its transcription factors (TF). Each replicon has a specific functional and evolutionary mark; which might also emerge from the analysis of their regulatory signatures. Here we have studied the plasticity of the regulatory network within and outside the S. meliloti species, looking for the presence of 41 TFs binding motifs in 51 strains and 5 related rhizobial species. We have detected a preference of several TFs for one of the three replicons, and the function of regulated genes was found to be in accordance with the overall replicon functional signature: house-keeping functions for the chromosome, metabolism for the chromid, symbiosis for the megaplasmid. This therefore suggests a replicon-specific wiring of the regulatory network in the S. meliloti species. At the same time a significant part of the predicted regulatory network is shared between the chromosome and the chromid, thus adding an additional layer by which the chromid integrates itself in the core genome. Furthermore, the regulatory network distance was found to be correlated with both promoter regions and accessory genome evolution inside the species, indicating that both pangenome compartments are involved in the regulatory network evolution. We also observed that genes which are not included in the species regulatory network are more likely to belong to the accessory genome, indicating that regulatory interactions should also be considered to predict gene conservation in bacterial pangenomes.

Molecular phylogeny of the nickel-resistance gene nreB and functional role in the nickel sensitive symbiotic nitrogen fixing bacterium Sinorhizobium meliloti

AimsHeavy-metal tolerance is a widespread phenotype in bacteria, particularly occurring in strains isolated from heavy-metal contaminated sites. Concerning nickel tolerance, the nre system is one of the most common. An ortholog of the nreB gene is present in the alfalfa symbiont Sinorhizobium meliloti also, which stir the attention on its functional role in such Ni-sensitive species and on the evolutionary relationships with Ni-resistant strain orthologs.MethodsPhylogenetic reconstruction and comparative genomics were performed to analyze the phylogenetic relationships of nreB orthologs, as well aa nreB deletion mutant S. meliloti strain was constructed and subjected to phenotypic analysis.ResultsPhylogenetic analysis of nreB genes indicated horizontal gene transfer events, possibly mediated via mobile genetic elements. Phenotype Microarray, biochemical and symbiotic analyses of the deletion mutant strain (ΔnreB) showed that in S. meliloti nreB is involved in the tolerance to several stresses other than Ni (mainly urea and copper), possibly partially mediated through the modulation of urease and hydrogenase activities.ConclusionsObtained results allowed us to speculate that nreB is a highly mobile gene cassette, spread in the bacterial phylogenetic tree via many HGT events, which could have been recruited to confer nickel-tolerance in strains thriving in contaminated environments, by small changes linked to its basic functions (e.g. modulation of urease and hydrogenase activity).

Permanent draft genome sequences of the symbiotic nitrogen fixing Ensifer meliloti strains BO21CC and AK58

Ensifer (syn. Sinorhizobium) meliloti is an important symbiotic bacterial species that fixes nitrogen. Strains BO21CC and AK58 were previously investigated for their substrate utilization and their plant-growth promoting abilities showing interesting features. Here, we describe the complete genome sequence and annotation of these strains. BO21CC and AK58 genomes are 6,985,065 and 6,974,333 bp long with 6,746 and 6,992 genes predicted, respectively.