Draft Genome Sequence of a Highly Virulent Strain of the Plant Pathogen Dickeya solani, IFB0099.

Dickeya solani is an important bacterial pathogen of potato cultivars in Europe. Here, we present the draft genome of D. solani strain IFB0099 isolated from potato in Poland that shows a high level of pectinolytic activity and a high virulence. This genome sequence is 5,094,121 bp and contains 4,365 protein-coding sequences.

StreamingTrim 1.0: a Java software for dynamic trimming of 16S rRNA sequence data from metagenetic studies.

Next-generation sequencing technologies are extensively used in the field of molecular microbial ecology to describe taxonomic composition and to infer functionality of microbial communities. In particular, the so-called barcode or metagenetic applications that are based on PCR amplicon library sequencing are very popular at present. One of the problems, related to the utilization of the data of these libraries, is the analysis of reads quality and removal (trimming) of low-quality segments, while retaining sufficient information for subsequent analyses (e.g. taxonomic assignment). Here, we present StreamingTrim, a DNA reads trimming software, written in Java, with which researchers are able to analyse the quality of DNA sequences in fastq files and to search for low-quality zones in a very conservative way. This software has been developed with the aim to provide a tool capable of trimming amplicon library data, retaining as much as taxonomic information as possible. This software is equipped with a graphical user interface for a user-friendly usage. Moreover, from a computational point of view, StreamingTrim reads and analyses sequences one by one from an input fastq file, without keeping anything in memory, permitting to run the computation on a normal desktop PC or even a laptop. Trimmed sequences are saved in an output file, and a statistics summary is displayed that contains the mean and standard deviation of the length and quality of the whole sequence file. Compiled software, a manual and example data sets are available under the BSD-2-Clause License at the GitHub repository at https://github.com/GiBacci/StreamingTrim/.

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. METHODS AND RESULTS: rpoE1 and nodC primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti. Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples. CONCLUSIONS: The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation. SIGNIFICANCE AND IMPACT OF THE STUDY: These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.

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 alpha-Proteobacteria representatives. In particular, cloned TRFs which discriminated between rhizosphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives.

Mediterranean Talitrus saltator (Crustacea, Amphipoda) as a biomonitor of heavy metals contamination.

The use of sandhoppers and beachfleas as biomonitors of heavy metals contamination is relatively recent. Using adult individuals of Talitrus saltator from nine localities on the northern Mediterranean Sea, we studied the concentrations of eight trace elements: Al, Cd, Cr, Fe, Hg, Pb, Cu, Zn, both in the substratum and in the individuals. We also carried out a preliminary investigation of the correspondence between the sandhoppers' genetic variability and heavy metal contamination at the sampling sites. T. saltator accumulated Cd, Cu, Zn and Hg (at higher concentrations than in the sand) and also Al and Fe (at lower concentrations than in the sand). It seems that Mediterranean sandhoppers do not accumulate Pb and Cr. An intraspecific comparison between northern European (Baltic) and Mediterranean populations of T. saltator was made. Finally, we observed a tendency to a positive correlation between the sandhoppers' genetic variability and heavy metals contamination.

Evolutionary dynamics of nickel hyperaccumulation in Alyssum revealed by its nrDNA analysis.

• Molecular phylogeny based on ribosomal internal transcribed spacer (ITS) sequences was studied to investigate the phyletic relationships among some nickel (Ni)-hyperaccumulating and nonhyperaccumulating species of the genus Alyssum in relation to their geographic distribution and Ni-hyperaccumulating phenotype. • Thirty-seven samples belonging to 32 taxa were analysed by sequencing the polymerase chain reaction-amplified ITS region and performing neighbor joining, maximum parsimony and maximum likelihood phylogenetic analyses. • The ITS region in the sampled species varied from 221 to 307 bp of ITS1 and from 194 to 251 bp of ITS2. A total of 765 characters was used to infer the phylogeny and the average nucleotide variation detected was 15.15%. • Nickel-hyperaccumulation could have been lost or acquired independently more than once during the speciation of the genus. The geographical location of species could not be related to phylogenetic affinities.

Chloroplast genetic diversity and biogeography in the serpentine endemic Ni-hyperaccumulator Alyssum bertolonii.

• Chloroplast microsatellites (cpSSR) were used to analyze the patterns of genetic variation within and among populations of the serpentine endemic, Alyssum bertolonii . • Thirty-five different chloroplast haplotypes were identified in 90 plants sampled from nine populations originating from the four disjunct northern-Italian serpentine regions where the species is found. • High levels of genetic diversity were found within each of the populations sampled. Analysis of Molecular Variance (AMOVA) showed high degrees of differentiation among both different populations of the same serpentine region and different regions (Φ ST = 0.622, Φ CT = 0.252, respectively). • The results indicated that: each population was established by few founders and then subsequently differentiated the existing chloroplast haplotypes; each population is a distinct genetic entity; and populations within the same serpentine region are more related than populations from different regions.

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.

Characterization of nickel-resistant bacteria isolated from serpentine soil.

In the present study, heterotrophic nickel-resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel-hyperaccumulating plant Alyssum bertolonii. Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant. The proportion of nickel-resistant cfu was higher in proximity to the plant than in free soil. A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy-metal resistant phenotypes. The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing. These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces. Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.

Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils.

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.

ISRm10: a new insertion sequence of Sinorhizobium meliloti: nucleotide sequence and geographic distribution.

In this study the description of a new insertion sequence of Sinorhizobium meliloti, ISRm10, is reported. ISRm10 was found in the intergenic region between nodJ and nodQ of a natural isolated strain. ISRm10 was 1047 bp long and showed the typical features of the ISs belonging to the IS630-Tc1/IS3 superfamily. In particular the ISRm10 nucleotide sequence showed the highest homology (62%) with a Sphingomonas aromaticivorans IS. ISRm10 was present in 32% of the analyzed S. meliloti strains while it was not found in the reference strains of other Rhizobium species.

Mutations of the Apc gene in experimental colorectal carcinogenesis induced by azoxymethane in F344 rats.

We investigated in the rat the role of the Apc gene, which is mutated in familial adenomatous polyposis and sporadic colon cancer in the process leading from normal colonic mucosa to aberrant crypt foci (ACF) and finally to adenomas and adenocarcinomas. We analysed mutations in exon 15 of the rat Apc gene using in vitro synthesized protein assay in 66 ACF and in 28 colon tumours induced by azoxymethane. No Apc mutations were found in ACF, whereas five mutations were found in the tumours. The data suggest that mutations of the Apc gene are associated with the transition from ACF to adenoma and adenocarcinoma and not from normal mucosa to ACF.

Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti strains by Medicago sativa.

We analysed the genetic diversity of 270 Sinorhizobium meliloti strains isolated from nodules of three different Medicago sativa varieties, planted in three different Italian soils, combining the Analysis of Molecular Variance (AMOVA) with the Random Amplified Polymorphic DNA (RAPD) technique to estimate variance among RAPD patterns with the aim to draw an objective description of the population genetic structure. Results indicated that a general intraspecific genetic diversity was globally distributed among all the population, however a very high level of diversity was found among strains nodulating different Medicago sativa varieties. Moreover the distribution of the RAPD haplotypes among the plant varieties also showed to be non-random. The overall data indicated that the plant genotype is a major factor in shaping the genetic structure of this natural Rhizobium population.

Duplication of insertion element IS50 associated with Tn5 transposition in Azospirillum brasilense.

The characterization of a DNA fragment with a Tn5 insertion in a regulatory nif gene of Azospirillum brasilense is reported. Restriction endonuclease mapping, Southern hybridization with a Tn5 probe, and nucleotide sequencing revealed that IS50 had duplicated in Tn5. The duplication of an IS50 element suggests the occurrence of a replicative mechanism of transposition. A strategy, based on the bacterial ability of homologous recombination that was used to precisely eliminate Tn5 along with the duplicated IS50 element, is presented.

Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.

Cloning, nucleotide sequencing, and expression of the Azospirillum brasilense lon gene: involvement in iron uptake.

The lon gene of Escherichia coli encodes the lon (La) protease, which is associated with cellular protein degradation. A lon gene homolog from Azospirillum brasilense, a nitrogen-fixing soil bacterium which lives in association with the roots of cereal grasses, was cloned and characterized. The nucleotide sequence of the A. brasilense lon gene was determined. It contains an open reading frame that encodes a protein of 810 amino acids with a predicted molecular mass of about 90 kDa. The deduced amino acid sequence showed a high level of homology with the sequences of all the known lon gene products. An open reading frame homologous to the E. coli clpX gene was found in front of the lon gene. Transcriptional analysis showed that the lon gene of A. brasilense is induced by heat shock and that the mRNA is monocistronic. An A. brasilense mutant, with Tn5 inserted in the lon gene, was shown to be defective in iron uptake and failed to express two membrane proteins that are induced by iron starvation in the parental strain.

Phylogeny of the genus Azospirillum based on 16S rDNA sequence.

The 16S rDNA of 17 strains of Azospirillum, 14 assigned to one of the known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum, and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the alpha subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.