Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation.

BACKGROUND: Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS). RESULTS: We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC≥1, P-value<0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications. CONCLUSIONS: Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.

Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data.

Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis.

Genomic analysis of four strains of Corynebacterium pseudotuberculosis bv. Equi isolated from horses showing distinct signs of infection.

The genomes of four strains (MB11, MB14, MB30, and MB66) of the species Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, completely assembled, and their gene content and structure were analyzed. The strains were isolated from horses with distinct signs of infection, including ulcerative lymphangitis, external abscesses on the chest, or internal abscesses on the liver, kidneys, and lungs. The average size of the genomes was 2.3 Mbp, with 2169 (Strain MB11) to 2235 (Strain MB14) predicted coding sequences (CDSs). An optical map of the MB11 strain generated using the KpnI restriction enzyme showed that the approach used to assemble the genome was satisfactory, producing good alignment between the sequence observed in vitro and that obtained in silico. In the resulting Neighbor-Joining dendrogram, the C. pseudotuberculosis strains sequenced in this study were clustered into a single clade supported by a high bootstrap value. The structural analysis showed that the genomes of the MB11 and MB14 strains were very similar, while the MB30 and MB66 strains had several inverted regions. The observed genomic characteristics were similar to those described for other strains of the same species, despite the number of inversions found. These genomes will serve as a basis for determining the relationship between the genotype of the pathogen and the type of infection that it causes.

GapBlaster-A Graphical Gap Filler for Prokaryote Genomes.

The advent of NGS (Next Generation Sequencing) technologies has resulted in an exponential increase in the number of complete genomes available in biological databases. This advance has allowed the development of several computational tools enabling analyses of large amounts of data in each of the various steps, from processing and quality filtering to gap filling and manual curation. The tools developed for gap closure are very useful as they result in more complete genomes, which will influence downstream analyses of genomic plasticity and comparative genomics. However, the gap filling step remains a challenge for genome assembly, often requiring manual intervention. Here, we present GapBlaster, a graphical application to evaluate and close gaps. GapBlaster was developed via Java programming language. The software uses contigs obtained in the assembly of the genome to perform an alignment against a draft of the genome/scaffold, using BLAST or Mummer to close gaps. Then, all identified alignments of contigs that extend through the gaps in the draft sequence are presented to the user for further evaluation via the GapBlaster graphical interface. GapBlaster presents significant results compared to other similar software and has the advantage of offering a graphical interface for manual curation of the gaps. GapBlaster program, the user guide and the test datasets are freely available at https://sourceforge.net/projects/gapblaster2015/. It requires Sun JDK 8 and Blast or Mummer.

GIPSy: Genomic island prediction software.

Bacteria are highly diverse organisms that are able to adapt to a broad range of environments and hosts due to their high genomic plasticity. Horizontal gene transfer plays a pivotal role in this genome plasticity and in evolution by leaps through the incorporation of large blocks of genome sequences, ordinarily known as genomic islands (GEIs). GEIs may harbor genes encoding virulence, metabolism, antibiotic resistance and symbiosis-related functions, namely pathogenicity islands (PAIs), metabolic islands (MIs), resistance islands (RIs) and symbiotic islands (SIs). Although many software for the prediction of GEIs exist, they only focus on PAI prediction and present other limitations, such as complicated installation and inconvenient user interfaces. Here, we present GIPSy, the genomic island prediction software, a standalone and user-friendly software for the prediction of GEIs, built on our previously developed pathogenicity island prediction software (PIPS). We also present four application cases in which we crosslink data from literature to PAIs, MIs, RIs and SIs predicted by GIPSy. Briefly, GIPSy correctly predicted the following previously described GEIs: 13 PAIs larger than 30kb in Escherichia coli CFT073; 1 MI for Burkholderia pseudomallei K96243, which seems to be a miscellaneous island; 1 RI of Acinetobacter baumannii AYE, named AbaR1; and, 1 SI of Mesorhizobium loti MAFF303099 presenting a mosaic structure. GIPSy is the first life-style-specific genomic island prediction software to perform analyses of PAIs, MIs, RIs and SIs, opening a door for a better understanding of bacterial genome plasticity and the adaptation to new traits.

Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon.

In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain PA01, Isolated from Sheep in Pará, Brazil.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis disease. In this work, we present the first complete genome sequence of Corynebacterium pseudotuberculosis strain PA01, isolated in northern Brazil from an infected sheep. The genome length is 2,337,920 bp, and 2,003 coding sequences (CDS), 12 rRNAs, and 49 tRNAs were predicted.

De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T. trichiura adult worm. Besides, orthologs of proteins demonstrated to have potent immunomodulatory properties in related parasitic helminths were also predicted from the T. trichiura de novo assembled transcriptome.

The impact of quality filter for RNA-Seq.

BACKGROUND: With the emergence of large-scale sequencing platforms since 2005, there has been a great revolution regarding methods for decoding DNA sequences, which have also affected quantitative and qualitative gene expression analyses through the RNA-Sequencing technique. However, issues related to the amount of data required for the analyses have been considered because they affect the reliability of the experiments. Thus, RNA depletion during sample preparation may influence the results. Moreover, because data produced by these platforms show variations in quality, quality filters are often used to remove sequences likely to contain errors to increase the accuracy of the results. However, when reads of quality filters are removed, the expression profile in RNA-Seq experiments may be influenced. RESULT: The present study aimed to analyze the impact of different quality filter values for Corynebacterium pseudotuberculosis (sequenced by SOLiD platform), Microcystis aeruginosa and Kineococcus radiotolerans (sequenced by Illumina platform) RNA-Seq data. Although up to 47.9% of the reads produced by the SOLiD technology were removed after the QV20 quality filter is applied, and 15.85% were removed from K. radiotolerans data set using the QV30 filter, Illumina data showed the largest number of unique differentially expressed genes after applying the most stringent filter (QV30), with 69 genes. In contrast, for SOLiD, the acid stress condition with the QV20 filter yielded only 41 unique differentially expressed genes. Even for the highest quality M. aeruginosa data, the quality filter affected the expression profile. The most stringent quality filter generated a greater number of unique differentially expressed genes: 9 for high molecular weight dissolved organic matter condition and 12 for low P conditions. CONCLUSION: Even high-accuracy sequencing technologies are subject to the influence of quality filters when evaluating RNA-Seq data using the reference approach.

Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses.

Corynebacterium pseudotuberculosis causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death (Ruiz et al., 2011) [1]. This bacterium was grown under osmotic (2 M), acid (pH) and heat (50 °C) stress and under control (Normal-BHI brain heart infusion) conditions, which simulate the conditions faced by the bacteria during the infectious process. Subsequently, cDNA of each condition was sequenced by the SOLiD3 Plus platform using the RNA-Seq technique [2], [3], [4]. The data produced was processed to evaluate the differential gene expression, which is helpful to understand the adaptation mechanisms during the infection in the host. The sequencing data of all conditions are available in the European Bioinformatics Institute (EBI) repository under accession number E-MTAB-2017.

Genome Sequence of Corynebacterium pseudotuberculosis MB20 bv. equi Isolated from a Pectoral Abscess of an Oldenburg Horse in California.

The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi.