Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi.

BACKGROUND: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. METHODS: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. RESULTS: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)]. CONCLUSIONS: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.

Corynebacterium pseudotuberculosis may be under anagenesis and biovar Equi forms biovar Ovis: a phylogenic inference from sequence and structural analysis.

BACKGROUND: Corynebacterium pseudotuberculosis can be classified into two biovars or biovars based on their nitrate-reducing ability. Strains isolated from sheep and goats show negative nitrate reduction and are termed biovar Ovis, while strains from horse and cattle exhibit positive nitrate reduction and are called biovar Equi. However, molecular evidence has not been established so far to understand this difference, specifically if these C. pseudotuberculosis strains are under an evolutionary process. RESULTS: The ERIC 1 + 2 Minimum-spanning tree from 367 strains of C. pseudotuberculosis showed that the great majority of biovar Ovis strains clustered together, but separately from biovar Equi strains that also clustered amongst themselves. Using evolutionarily conserved genes (rpoB, gapA, fusA, and rsmE) and their corresponding amino acid sequences, we analyzed the phylogenetic relationship among eighteen strains of C. pseudotuberculosis belonging to both biovars Ovis and Equi. Additionally, conserved point mutation based on structural variation analysis was also carried out to elucidate the genotype-phenotype correlations and speciation. We observed that the biovars are different at the molecular phylogenetic level and a probable anagenesis is occurring slowly within the species C. pseudotuberculosis. CONCLUSIONS: Taken together the results suggest that biovar Equi is forming the biovar Ovis. However, additional analyses using other genes and other bacterial strains are required to further support our anagenesis hypothesis in C. pseudotuberculosis.

DISMIRA: Prioritization of disease candidates in miRNA-disease associations based on maximum weighted matching inference model and motif-based analysis.

BACKGROUND: MicroRNAs (miRNAs) have increasingly been found to regulate diseases at a significant level. The interaction of miRNA and diseases is a complex web of multilevel interactions, given the fact that a miRNA regulates upto 50 or more diseases and miRNAs/diseases work in clusters. The clear patterns of miRNA regulations in a disease are still elusive. METHODS: In this work, we approach the miRNA-disease interactions from a network scientific perspective and devise two approaches - maximum weighted matching model (a graph theoretical algorithm which provides the result by solving an optimization equation of selecting the most prominent set of diseases) and motif-based analyses (which investigates the motifs of the miRNA-disease network and selects the most prominent set of diseases based on their maximum number of participation in motifs, thereby revealing the miRNA-disease interaction dynamics) to determine and prioritize the set of diseases which are most certainly impacted upon the activation of a group of queried miRNAs, in a miRNA-disease network. RESULTS AND CONCLUSION: Our tool, DISMIRA implements the above mentioned approaches and presents an interactive visualization which helps the user in exploring the networking dynamics of miRNAs and diseases by analyzing their neighbors, paths and topological features. A set of miRNAs can be used in this analysis to get the associated diseases for the input group of miRs with ranks and also further analysis can be done to find key miRs or diseases, shortest paths etc. DISMIRA can be accessed online for free at http://bnet.egr.vcu.edu:8080/dismira.

CMRegNet-An interspecies reference database for corynebacterial and mycobacterial regulatory networks.

BACKGROUND: Organisms utilize a multitude of mechanisms for responding to changing environmental conditions, maintaining their functional homeostasis and to overcome stress situations. One of the most important mechanisms is transcriptional gene regulation. In-depth study of the transcriptional gene regulatory network can lead to various practical applications, creating a greater understanding of how organisms control their cellular behavior. DESCRIPTION: In this work, we present a new database, CMRegNet for the gene regulatory networks of Corynebacterium glutamicum ATCC 13032 and Mycobacterium tuberculosis H37Rv. We furthermore transferred the known networks of these model organisms to 18 other non-model but phylogenetically close species (target organisms) of the CMNR group. In comparison to other network transfers, for the first time we utilized two model organisms resulting into a more diverse and complete network of the target organisms. CONCLUSION: CMRegNet provides easy access to a total of 3,103 known regulations in C. glutamicum ATCC 13032 and M. tuberculosis H37Rv and to 38,940 evolutionary conserved interactions for 18 non-model species of the CMNR group. This makes CMRegNet to date the most comprehensive database of regulatory interactions of CMNR bacteria. The content of CMRegNet is publicly available online via a web interface found at http://lgcm.icb.ufmg.br/cmregnet .

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii.

BACKGROUND: Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost. RESULTS: 21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources. CONCLUSION: These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Proteome scale comparative modeling for conserved drug and vaccine targets identification in Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

Genomic decoding of Theobroma grandiflorum (cupuassu) at chromosomal scale: evolutionary insights for horticultural innovation.

BACKGROUND: Theobroma grandiflorum (Malvaceae), known as cupuassu, is a tree indigenous to the Amazon basin, valued for its large fruits and seed pulp, contributing notably to the Amazonian bioeconomy. The seed pulp is utilized in desserts and beverages, and its seed butter is used in cosmetics. Here, we present the sequenced telomere-to-telomere genome of cupuassu, disclosing its genomic structure, evolutionary features, and phylogenetic relationships within the Malvaceae family. FINDINGS: The cupuassu genome spans 423 Mb, encodes 31,381 genes distributed in 10 chromosomes, and exhibits approximately 65% gene synteny with the Theobroma cacao genome, reflecting a conserved evolutionary history, albeit punctuated with unique genomic variations. The main changes are pronounced by bursts of long-terminal repeat retrotransposons at postspecies divergence, retrocopied and singleton genes, and gene families displaying distinctive patterns of expansion and contraction. Furthermore, positively selected genes are evident, particularly among retained and dispersed tandem and proximal duplicated genes associated with general fruit and seed traits and defense mechanisms, supporting the hypothesis of potential episodes of subfunctionalization and neofunctionalization following duplication, as well as impact from distinct domestication process. These genomic variations may underpin the differences observed in fruit and seed morphology, ripening, and disease resistance between cupuassu and the other Malvaceae species. CONCLUSIONS: The cupuassu genome offers a foundational resource for both breeding improvement and conservation biology, yielding insights into the evolution and diversity within the genus Theobroma.

Genome sequence of Staphylococcus aureus Newbould 305, a strain associated with mild bovine mastitis.

Staphylococcus aureus is a major etiological agent of mastitis in ruminants. We report here the genome sequence of bovine strain Newbould 305, isolated in the 1950s in a case of bovine mastitis and now used as a model strain able to reproducibly induce chronic mastitis in cows.

Genome sequence of the Corynebacterium pseudotuberculosis Cp316 strain, isolated from the abscess of a Californian horse.

The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse.

Complete genome sequence of Corynebacterium pseudotuberculosis Cp31, isolated from an Egyptian buffalo.

Corynebacterium pseudotuberculosis is of major veterinary importance because it affects many animal species, causing economically significant livestock diseases and losses. Therefore, the genomic sequencing of various lines of this organism, isolated from different hosts, will aid in the development of diagnostic methods and new prevention and treatment strategies and improve our knowledge of the biology of this microorganism. In this study, we present the genome of C. pseudotuberculosis Cp31, isolated from a buffalo in Egypt.

Genome sequence of Exiguobacterium antarcticum B7, isolated from a biofilm in Ginger Lake, King George Island, Antarctica.

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e.g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.

Whole-genome sequence of Corynebacterium pseudotuberculosis strain Cp162, isolated from camel.

Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Corynebacterium pseudotuberculosis strain Cp162, collected from the external neck abscess of a camel in the United Kingdom.

Complete genome sequence of Corynebacterium pseudotuberculosis strain Cp267, isolated from a llama.

In this work we report the genome of Corynebacterium pseudotuberculosis strain 267, isolated from a llama. This pathogen is of great veterinary and economic importance, as it is the cause of caseous lymphadenitis in several livestock species around the world and causes significant losses due to the high cost of treatment.

Complete genome sequence of Corynebacterium pseudotuberculosis strain 1/06-A, isolated from a horse in North America.

Corynebacterium pseudotuberculosis causes disease in several animal species, although distinct biovars exist that appear to be restricted to specific hosts. In order to facilitate a better understanding of the differences between biovars, we report here the complete genome sequence of the equine pathogen Corynebacterium pseudotuberculosis strain 1/06-A.

Complete genome sequences of Corynebacterium pseudotuberculosis strains 3/99-5 and 42/02-A, isolated from sheep in Scotland and Australia, respectively.

Here, we report the whole-genome sequences of two ovine-pathogenic Corynebacterium pseudotuberculosis isolates: strain 3/99-5, which represents the first C. pseudotuberculosis genome originating from the United Kingdom, and 42/02-A, the second from Australia. These genome sequences will contribute to the objective of determining the global pan-genome of this bacterium.

The Corynebacterium pseudotuberculosis in silico predicted pan-exoproteome.

BACKGROUND: Pan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results. RESULTS: The complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes. CONCLUSIONS: The in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.

Complete genome sequence of Corynebacterium pseudotuberculosis I19, a strain isolated from a cow in Israel with bovine mastitis.

This work reports the completion and annotation of the genome sequence of Corynebacterium pseudotuberculosis I19, isolated from an Israeli dairy cow with severe clinical mastitis. To present the whole-genome sequence, a de novo assembly approach using 33 million short (25-bp) mate-paired SOLiD reads only was applied. Furthermore, the automatic, functional, and manual annotations were attained with the use of several algorithms in a multistep process.

Whole-genome sequence of Corynebacterium pseudotuberculosis PAT10 strain isolated from sheep in Patagonia, Argentina.

In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.

Complete genome sequence of type strain Campylobacter fetus subsp. venerealis NCTC 10354T.

Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354(T) are reported.