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

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 p SymB 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. © 2013 The Author(s)

Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network.

BACKGROUND: Phylogenetic methods are well-established bioinformatic tools for sequence analysis, allowing to describe the non-independencies of sequences because of their common ancestor. However, the evolutionary profiles of bacterial genes are often complicated by hidden paralogy and extensive and/or (multiple) horizontal gene transfer (HGT) events which make bifurcating trees often inappropriate. In this context, plasmid sequences are paradigms of network-like relationships characterizing the evolution of prokaryotes. Actually, they can be transferred among different organisms allowing the dissemination of novel functions, thus playing a pivotal role in prokaryotic evolution. However, the study of their evolutionary dynamics is complicated by the absence of universally shared genes, a prerequisite for phylogenetic analyses. RESULTS: To overcome such limitations we developed a bioinformatic package, named Blast2Network (B2N), allowing the automatic phylogenetic profiling and the visualization of homology relationships in a large number of plasmid sequences. The software was applied to the study of 47 completely sequenced plasmids coming from Escherichia, Salmonella and Shigella spps. CONCLUSION: The tools implemented by B2N allow to describe and visualize in a new way some of the evolutionary features of plasmid molecules of Enterobacteriaceae; in particular it helped to shed some light on the complex history of Escherichia, Salmonella and Shigella plasmids and to focus on possible roles of unannotated proteins.The proposed methodology is general enough to be used for comparative genomic analyses of bacteria.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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

<p>Abstract</p> <p>Background</p> <p>In the bacterium <it>Caulobacter crescentus</it>, 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 <it>Caulobacter</it>, its conservation and functionality in the other alpha-proteobacteria are still poorly understood.</p> <p>Results</p> <p>Orthologs of <it>Caulobacter </it>factors involved in the regulation of CtrA were systematically scanned in genomes of alpha-proteobacteria. In particular, orthologous genes of the <it>divL-cckA-chpT-ctrA </it>phosphorelay, the <it>divJ</it>-<it>pleC</it>-<it>divK </it>two-component system, the <it>cpdR</it>-<it>rcdA</it>-<it>clpPX </it>proteolysis system, the methyltransferase <it>ccrM </it>and transcriptional regulators <it>dnaA </it>and <it>gcrA </it>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.</p> <p>Conclusions</p> <p>The 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 <it>Caulobacter </it>arranged in a variety of connections. Two schemes appeared to evolve: a complex circuit in <it>Caulobacterales </it>and <it>Rhizobiales </it>and a simpler one found in <it>Rhodobacterales</it>.</p

Genetic diversity and salt tolerance of bacterial communities from two Tunisian soils

Microbial ecology studies on arid soils are particularly important for the analysis of biological functions during desertification. Although much is known about the arid saline flora, few researches have directly compared the bacterial communities of saline arid soils with cultivated soils in Northern Africa. Bacterial communities present in two soils from Soliman (north of Tunisia), one salty and neglected, and the other cultivated, were investigated by using both cultivation dependent and independent approaches. The first approach was used to assess the presence of salt tolerant bacteria and the relationships among salt (NaCl) resistance phenotype, soil characteristics and phylogenetic assignment of strains. Total community analysis, performed by T-RFLP on total DNA, was carried out to investigate the relationships between total community fingerprinting with cultivated isolates diversity. The cultivated isolates from salty soil were more genetically diverse, harbouring strains that can grow at high salt concentration. Moreover, the salt resistance of isolates was found not to be related to any particular phylogenetic group, being widespread among isolates belonging to different bacterial subdivisions. Ribotype richness, evaluated as number of different T-RFLP bands (TRFs), was shown to be higher in the agricultural soil than in the salty soil and several agricultural soil-specific TRFs were detected

Role and regulation of ACC deaminase gene in Sinorhizobium meliloti: is it a symbiotic, rhizospheric or endophytic gene?

Plant-associated bacteria exhibit a number of different strategies and specific genes allow bacteria to communicate and metabolically interact with plant tissues. Among the genes found in the genomes of plant-associated bacteria, the gene encoding the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) is one of the most diffused. This gene is supposed to be involved in the cleaving of plant-produced ACC, the precursor of the plant stress-hormone ethylene toning down the plant response to infection. However, few reports are present on the actual role in rhizobia, one of the most investigated groups of plant-associated bacteria. In particular, still unclear is the origin and the role of acdS in symbiotic competitiveness and on the selective benefit it may confer to plant symbiotic rhizobia. Here we present a phylogenetic and functional analysis of acdS orthologs in the rhizobium model-species Sinorhizobium meliloti. Results showed that acdS orthologs present in S. meliloti pangenome have polyphyletic origin and likely spread through horizontal gene transfer, mediated by mobile genetic elements. When acdS ortholog from AK83 strain was cloned and assayed in S. meliloti 1021 (lacking acdS), no modulation of plant ethylene levels was detected, as well as no increase in fitness for nodule occupancy was found in the acdS-derivative strain compared to the parental one. Surprisingly, AcdS was shown to confer the ability to utilize formamide and some dipeptides as sole nitrogen source. Finally, acdS was shown to be negatively regulated by a putative leucine-responsive regulator (LrpL) located upstream to acdS sequence (acdR). acdS expression was induced by root exudates of both legumes and non-leguminous plants. We conclude that acdS in S. meliloti is not directly related to symbiotic interaction, but it could likely be involved in the rhizospheric colonization or in the endophytic behavior

Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains

BACKGROUND: Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This species occupies different ecological niches, being present as a free-living soil bacterium and as a symbiont of plant root nodules. The genome of the type strain Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6 Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide microarrays to estimate genetic variation at the genomic level in four natural strains, two isolated from Italian agricultural soil and two from desert soil in the Aral Sea region. RESULTS: From 4.6 to 5.7 percent of the genes showed a pattern of hybridisation concordant with deletion, nucleotide divergence or ORF duplication when compared to the type strain Rm 1021. A large number of these polymorphisms were confirmed by sequencing and Southern blot. A statistically significant fraction of these variable genes was found on the pSymA megaplasmid and grouped in clusters. These variable genes were found to be mainly transposases or genes with unknown function. CONCLUSION: The obtained results allow to conclude that the symbiosis-required megaplasmid pSymA can be considered the major hot-spot for intra-specific differentiation in S. meliloti

Genetic diversity of dinitrogen-fixing bacterial communities in soil amended with olive husks

The industrial production of olive oil is accompanied by the accumulation of large quantities of by-products from the olive milling industry that are commonly dispersed as fertilisers, which are nowadays suspected to have potential toxic effects on is omicroflora. The aim of this work has been the investigation of the genetic diversity of bacterial communities present in soil treated with olive husks focusing on the dinitrogen-fixing bacteria.nifH genes were amplified from total soil DNA using universal primers, cloned and typed by restriction analysis and sequencing of representative haplotypes. On the same samples, DGGE analysis on amplified 16S rDNA was performed aiming at monitoring modifications in the total community pattern. Results showed a high genetic diversity ofnifH genes within the community, which was well in agreement with the total community profiles obtained by DGGE on 16SrDNA. Most of thenifH gene fragments (19 out of 32) were found to be similar to sequences related with clostridia