Emanuele G. Biondi

CONTIGuator: a bacterial genomes finishing tool for structural insights on draft genomes.

Recent developments in sequencing technologies have given the opportunity to sequence many bacterial genomes with limited cost and labor, compared to previous techniques. However, a limiting step of genome sequencing is the finishing process, needed to infer the relative position of each contig and close sequencing gaps. An additional degree of complexity is given by bacterial species harboring more than one replicon, which are not contemplated by the currently available programs. The availability of a large number of bacterial genomes allows geneticists to use complete genomes (possibly from the same species) as templates for contigs mapping.Here we present CONTIGuator, a software tool for contigs mapping over a reference genome which allows the visualization of a map of contigs, underlining loss and/or gain of genetic elements and permitting to finish multipartite genomes. The functionality of CONTIGuator was tested using four genomes, demonstrating its improved performances compared to currently available programs.Our approach appears efficient, with a clear visualization, allowing the user to perform comparative structural genomics analysis on draft genomes. CONTIGuator is a Python script for Linux environments and can be used on normal desktop machines and can be downloaded from http://contiguator.sourceforge.net.

The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis

BackgroundIn the bacterium Caulobacter crescentus, CtrA coordinates DNA replication, cell division, and polar morphogenesis and is considered the cell cycle master regulator. CtrA activity varies during cell cycle progression and is modulated by phosphorylation, proteolysis and transcriptional control. In a phosphorylated state, CtrA binds specific DNA sequences, regulates the expression of genes involved in cell cycle progression and silences the origin of replication. Although the circuitry regulating CtrA is known in molecular detail in Caulobacter, its conservation and functionality in the other alpha-proteobacteria are still poorly understood.ResultsOrthologs of Caulobacter factors involved in the regulation of CtrA were systematically scanned in genomes of alpha-proteobacteria. In particular, orthologous genes of the divL-cckA-chpT-ctrA phosphorelay, the divJ-pleC-divK two-component system, the cpdR-rcdA-clpPX proteolysis system, the methyltransferase ccrM and transcriptional regulators dnaA and gcrA were identified in representative genomes of alpha-proteobacteria. CtrA, DnaA and GcrA binding sites and CcrM putative methylation sites were predicted in promoter regions of all these factors and functions controlled by CtrA in all alphas were predicted.ConclusionsThe regulatory cell cycle architecture was identified in all representative alpha-proteobacteria, revealing a high diversification of circuits but also a conservation of logical features. An evolutionary model was proposed where ancient alphas already possessed all modules found in Caulobacter arranged in a variety of connections. Two schemes appeared to evolve: a complex circuit in Caulobacterales and Rhizobiales and a simpler one found in Rhodobacterales.

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.

Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.

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.

Genetic Diversity of Bacterial Communities of Serpentine Soil and of Rhizosphere of the Nickel-Hyperaccumulator Plant Alyssum bertolonii

Serpentine soils are characterized by high levels of heavy metals (Ni, Co, Cr), and low levels of important plant nutrients (P, Ca, N). Because of these inhospitable edaphic conditions, serpentine soils are typically home to a very specialized flora including endemic species as the nickel hyperaccumulator Alyssum bertolonii. Although much is known about the serpentine flora, few researches have investigated the bacterial communities of serpentine areas. In the present study bacterial communities were sampled at various distances from A. bertolonii roots in three different serpentine areas and their genetic diversity was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The obtained results indicated the occurrence of a high genetic diversity and heterogeneity of the bacterial communities present in the different serpentine areas. Moreover, TRFs (terminal restriction fragments) common to all the investigated A. bertolonii rhizosphere samples were found. A new cloning strategy was applied to 27 TRFs that were sequenced and taxonomically interpreted as mainly belonging to Gram-positive and α-Proteobacteria representatives. In particular, cloned TRFs which discriminated between rhizosphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives.

DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

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.

Metabolic Capacity of Sinorhizobium (Ensifer) meliloti Strains as Determined by Phenotype MicroArray Analysis

ABSTRACT Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The high genetic diversity of its natural populations has been the subject of extensive analysis. Recent genomic studies of several isolates revealed a high content of variable genes, suggesting a correspondingly large phenotypic differentiation among strains of S. meliloti. Here, using the Phenotype MicroArray (PM) system, hundreds of different growth conditions were tested in order to compare the metabolic capabilities of the laboratory reference strain Rm1021 with those of four natural S. meliloti isolates previously analyzed by comparative genomic hybridization (CGH). The results of PM analysis showed that most phenotypic differences involved carbon source utilization and tolerance to osmolytes and pH, while fewer differences were scored for nitrogen, phosphorus, and sulfur source utilization. Only the variability of the tested strain in tolerance to sodium nitrite and ammonium sulfate of pH 8 was hypothesized to be associated with the genetic polymorphisms detected by CGH analysis. Colony and cell morphologies and the ability to nodulate Medicago truncatula plants were also compared, revealing further phenotypic diversity. Overall, our results suggest that the study of functional (phenotypic) variability of S. meliloti populations is an important and complementary step in the investigation of genetic polymorphism of rhizobia and may help to elucidate rhizobial evolutionary dynamics, including adaptation to diverse environments.

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.