Alessio Mengoni

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.

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.

Isolation and Characterization of Endophytic Bacteria from the Nickel Hyperaccumulator Plant Alyssum bertolonii

We report the isolation and characterization of endophytic bacteria, endemic to serpentine outcrops of Central Italy, from a nickel hyperaccumulator plant, Alyssum bertolonii Desv. (Brassicaceae). Eighty-three endophytic bacteria were isolated from roots, stems, and leaves of A. bertolonii and classified by restriction analysis of 16S rDNA (ARDRA) and partial 16S rDNA sequencing in 23 different taxonomic groups. All isolates were then screened for siderophore production and for resistance to heavy metals. One isolate representative of each ARDRA group was then tested for plant tissue colonization ability in sterile culture.Obtained results pointed out that, despite the high concentration of heavy metals present in its tissues, A. bertolonii harbors an endophytic bacterial flora showing a high genetic diversity as well as a high level of resistance to heavy metals that could potentially help plant growth and Ni hyperaccumulation.

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.

Genetic Diversity and Dynamics of Sinorhizobium meliloti Populations Nodulating Different Alfalfa Cultivars in Italian Soils

ABSTRACT We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.

Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L. (Caryophyllaceae): a random amplified polymorphic DNA analysis

Metal‐contaminated sites can occur naturally in serpentine outcrops or as consequence of anthropogenic activities, such as mining deposits, aerial fallout from smelters and industrial processes. Serpentine outcrops are characterized by high levels of nickel, cobalt and chromium and present a typical vegetation which includes endemisms and plants which also live in uncontaminated soils. These latter metal‐tolerant populations provide the opportunity to investigate the first steps in the differentiation of plant populations under severe selection pressure and to select plants to be used in the phytoremediation of industrially contaminated soils. In this report eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in noncontaminated soil in central Italy, were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the pattern of genetic variation. The genetic diversity observed in populations at copper mine deposits was found to be at least as high as that of the neighbouring serpentine populations. Analysis of molecular variance (amova) of the RAPD markers gave high statistical significance to the groupings of populations according: (i) with their geographical location; and (ii) with the metals present in the soil of origin (copper vs. nickel), indicating that RAPD markers detected a polymorphism related to the soil contamination by copper. Finally, two RAPD bands exclusive to copper‐tolerant populations were identified.

Fluctuation of bacteria isolated from elm tissues during different seasons and from different plant organs

In this work we isolated a culturable endophytic aerobic heterotrophic bacterial community from the stem and root tissues of elm trees (Ulmus spp.) and analyzed its fluctuations. A total of 724 bacterial isolates were collected at different times (April, June, September and December) from two elm trees, one infected with Elm Yellows phytoplasmas, and one which was healthy-looking. The isolates were grouped into 82 haplotypes, identified by means of amplified ribosomal DNA restriction analysis (ARDRA) using the restriction enzyme AluI, suggesting that the genetic diversity of the bacterial community was very high. The taxonomic position of the isolates belonging to the twelve main haplotypes, representing more than 72% of the total population, was determined by 16S rDNA sequencing. The main genera were Bacillus, Curtobacterium, Pseudomonas, Stenotrophomonas, Sphingomonas, Enterobacter, and Staphylococcus. The fluctuations in the bacterial community, determined by different parameters (seasonal changes, plant organ, presence of phytoplasmas) were studied, revealing that they were influenced both by variations in temperature (warm or cold according to the season) and by the organ examined (roots or stems). The role of the phytopathogenic status in these fluctuations was also discussed.

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 diversity of heavy metal‐tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model‐based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper‐tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (amova) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal‐tolerant populations.

Exploring the evolutionary dynamics of plasmids: the Acinetobacter pan-plasmidome

BackgroundProkaryotic plasmids have a dual importance in the microbial world: first they have a great impact on the metabolic functions of the host cell, providing additional traits that can be accumulated in the cell without altering the gene content of the bacterial chromosome. Additionally and/or alternatively, from a genome perspective, plasmids can provide a basis for genomic rearrangements via homologous recombination and so they can facilitate the loss or acquisition of genes during these events, which eventually may lead to horizontal gene transfer (HGT). Given their importance for conferring adaptive traits to the host organisms, the interest in plasmid sequencing is growing and now many complete plasmid sequences are available online.ResultsBy using the newly developed Blast2Network bioinformatic tool, a comparative analysis was performed on the plasmid and chromosome sequence data available for bacteria belonging to the genus Acinetobacter, an ubiquitous and clinically important group of γ-proteobacteria. Data obtained showed that, although most of the plasmids lack mobilization and transfer functions, they have probably a long history of rearrangements with other plasmids and with chromosomes. Indeed, traces of transfers between different species can be disclosed.ConclusionsWe show that, by combining plasmid and chromosome similarity, identity based, network analysis, an evolutionary scenario can be described even for highly mobile genetic elements that lack extensively shared genes. In particular we found that transposases and selective pressure for mercury resistance seem to have played a pivotal role in plasmid evolution in Acinetobacter genomes sequenced so far.