Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of Gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.     

Phylogenetic diversity of bacteria isolates from the Qinghai-Tibet Plateau permafrost region

The Qinghai-Tibet Plateau in east Asia is a unique and important permafrost environment. However, its microbiology remains largely unexplored to date. In this study, sediment samples were collected from the Qinghai-Tibet Plateau permafrost region, bacteria isolation procedures were performed 8 times, and the samples incubated at 4 degrees C for nearly 3 months. The number of colony forming units (cfu) ranged from 0 to 10(7)/(g dry soil). The quantity of culturable bacteria grew exponentially within the first few weeks, and then slowed gradually to a plateau. Phylogenetic analyses indicated that all the isolates fell into 6 categories: high G+C Gram-positive bacteria, low G+C Gram-positive bacteria, alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, and Cytophaga-Flavobacterium-Bacteroides group bacteria. The isolates belong to 19 genera, but the genera Arthrobacter and Pseudomonas were predominant. With the increase in incubation time, the isolated populations changed in terms of both species and their respective quantities. Of the 33 analyzed isolates, 9 isolates related to 8 genera might be new taxa. These results suggest that the Qinghai-Tibet Plateau permafrost region is a specific ecologic niche that accommodates an original microbial assemblage.     

Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates

Trichoderma harzianum is the collective name of a set of asexual fungal strains which exhibit heterogeneity in genome structure, DNA sequence and behavior. Contour-clamped homogeneous field (CHEF) electrophoresis of the chromosomes of ten isolates of T. harzianum revealed six clearly distinct electrophoretic karyotypes. Of the ten isolates analyzed, four (GH12, G109, Y and YF) could be classified in a single group with identical karyotypes, while the strains T35 and 315 formed a second group. The genome size characteristic of the different isolates fell into a broad range varying from 29.6 to 56.1?Mb. Gene assignments to the resolved chromosomes showed that all genes analyzed were localized on equivalent chromosomes in the isolates belonging to the same group. Analysis of randomly amplified polymorphic DNAs from the ten isolates confirmed the classification into groups and allowed us to distinguish between isolates T35 and 315, as well as between isolates GH12, G109, Y and YF. Direct confrontation assays using isolates of the same group showed compatible interactions, whereas the same experiment carried out with isolates of different groups showed an incompatible interaction characterized by an area of cell damage. Microscopic observation of the compatible interactions showed hyphal fusions between the isolates, similar to those described for vegetative compatible groups in other fungi. The molecular karyotypes correlated well with the compatibility of the isolates. In addition, we have evaluated both electrophoretic karyotype and randomly amplified polymorphic DNAs analysis as criteria for grouping isolates within the genus according to their capacity for biocontrol of plant pathogens.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

Molecular and functional characterization of O antigen transfer in Vibrio cholerae

The majority of Gram-negative bacteria transfer O antigen polysaccharides onto the lipid A-core oligosaccharide via the action of surface polymer:lipid A-core ligases (WaaL). Here, we characterize the WaaL proteins of Vibrio cholerae with emphasis on structural and functional characterization of O antigen transfer and core oligosaccharide recognition. We demonstrate that the activity of two distantly related O antigen ligases is dependent on the presence of N-acetylglucosamine, and substitution of an additional sugar, i.e. galactose, alters the site specificity of the core oligosaccharide necessitating discriminative WaaL types. Protein topology analysis and a conserved domain search identified two distinct conserved motifs in the periplasmic domains of WaaL proteins. Site-directed mutagenesis of the two motifs, shown for WaaLs of V. cholerae and Salmonella enterica, caused a loss of O antigen transfer activity. Moreover, analogy of topology and motifs between WaaLs and O polysaccharide polymerases (Wzy) reveals a relationship between the two protein families, suggesting that the catalyzed reactions are related to each other.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.     

Chemical de‐O‐glycosylation of glycoproteins for application in LC‐based proteomics

We describe a cyclic on‐column procedure for the sequential degradation of complex O‐glycans on proteins or peptides by periodate oxidation of sugars and cleavage of oxidation products by elimination. Desialylated glycoproteins were immobilized to alkali‐stable, reversed‐phase Poros 20 beads followed by two degradation cycles and the eluted apoproteins were either separated by SDS gel electrophoresis or digested with trypsin prior to LC/ESI‐MS. We demonstrate on the peptide and protein level that even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration or by racemization of the Lys/Arg residues. The protocol is applicable on gel‐immobilized glycoproteins after SDS gel electrophoresis. Conversion of O‐glycoproteins into their corresponding apoproteins should result in facilitated accessibility of tryptic cleavage sites, increase the numbers of peptide fragments, and accordingly enhance protein coverage and identification rates within the subproteome of mucin‐type O‐glycoproteins.     

Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli

Fifty-one isolates of Rhizobium leguminosarum biovar phaseoli from various geographic and ecological sources, largely in Mexico, were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 46 distinctive multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at the enzyme loci. Mean genetic diversity per enzyme locus among the 46 ETs was 0.691, the highest value yet recorded for any species of bacterium. The occurrence of strong nonrandom associations of alleles over loci suggested a basically clonal population structure, reflecting infrequent recombination of chromosomal genes. Multilocus genotypic diversity was unusually high, with the most strongly differentiated pairs of ETs having distinctive alleles at all 15 loci and major clusters of ETs diverging at genetic distances as large as 0.89. This great diversity in the chromosomal genome raises the possibility that R. leguminosarum biovar phaseoli is a polyphyletic assemblage of strains. As other workers have suggested, the inclusion of all strains capable of nodulating beans in a single biovar or species is genetically unrealistic and taxonomically misleading. A biologically meaningful classification of Rhizobium spp. should be based on assessment of variation in the chromosomal genome rather than on phenotypic characters, especially those mediated for the most part or wholly by plasmid-borne genes, such as host relationships.     

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.     

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates

AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.     

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Δvsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δvsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δvsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.     

Genetic diversity among Brazilian isolates of Beauveria bassiana: comparisons with non-Brazilian isolates and other Beauveria species

Aims:  The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp.
Methods and Results:  The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp.
Conclusion:  Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil.
Significance and Impact of the Study:  This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu . The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.     

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O‐formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20^oC) induces N‐formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N‐formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below –20^oC.     

Conservation and diversity among the three-dimensional folds of the Dicistroviridae intergenic region IRESes

Internal ribosome entry site (IRES) RNAs are necessary for successful infection of many pathogenic viruses, but the details of the RNA structure-based mechanism used to bind and manipulate the ribosome remain poorly understood. The IRES RNAs from the Dicistroviridae intergenic region (IGR) are an excellent model system to understand the fundamental tenets of IRES function, requiring no protein factors to manipulate the ribosome and initiate translation. Here, we explore the architecture of four members of the IGR IRESes, representative of the two divergent classes of these IRES RNAs. Using biochemical and structural probing methods, we show that despite sequence variability they contain a common three-dimensional fold. The three-dimensional architecture of the ribosome binding domain from these IRESes is organized around a core helical scaffold, around which the rest of the RNA molecule folds. However, subtle variation in the folds of these IRESes and the presence of an additional secondary structure element suggest differences in the details of their manipulation of the large ribosomal subunit. Overall, the results demonstrate how a conserved three-dimensional RNA fold governs ribosome binding and manipulation.