Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

CDH1 mutations in gastric cancer patients from northern Brazil identified by Next- Generation Sequencing (NGS).

Gastric cancer is considered to be the fifth highest incident tumor worldwide and the third leading cause of cancer deaths. Developing regions report a higher number of sporadic cases, but there are only a few local studies related to hereditary cases of gastric cancer in Brazil to confirm this fact. CDH1 germline mutations have been described both in familial and sporadic cases, but there is only one recent molecular description of individuals from Brazil. In this study we performed Next Generation Sequencing (NGS) to assess CDH1 germline mutations in individuals who match the clinical criteria for Hereditary Diffuse Gastric Cancer (HDGC), or who exhibit very early diagnosis of gastric cancer. Among five probands we detected CDH1 germline mutations in two cases (40%). The mutation c.1023T > G was found in a HDGC family and the mutation c.1849G > A, which is nearly exclusive to African populations, was found in an early-onset case of gastric adenocarcinoma. The mutations described highlight the existence of gastric cancer cases caused by CDH1 germline mutations in northern Brazil, although such information is frequently ignored due to the existence of a large number of environmental factors locally. Our report represent the first CDH1 mutations in HDGC described from Brazil by an NGS platform.

A Protocol for mtGenome Analysis on Large Sample Numbers.

The mitochondrial genome is widely studied in a variety of fields, such as population, forensic, and human and medical genetics. Most studies have been limited to a small portion of the sequence that, although highly diverse, does not describe the total variability. The arrival of modern high-throughput sequencing technologies has made it possible to investigate larger sequences in a shorter amount of time as well as in a more affordable fashion. This work aims to describe a protocol for sequencing and analyzing the complete mitochondrial genome with the Ion PGM™ platform. To evaluate the protocol, the mitochondrial genome was sequenced to approximately 210 Mbp, with high-quality sequences distributed between 12 samples that had an average coverage of 1023× per sample. Several variant callers were compared to improve the protocol outcome. The results suggest that it is possible to run up to 120 samples per run without any loss of any significant quality. Therefore, this protocol is an efficient and accurate tool for full mitochondrial genome analysis.

Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

BACKGROUND: The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium 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. In the current study, we simulated the conditions experienced by the bacteria during host infection. By sequencing transcripts using the SOLiDTM 3 Plus platform, we identified new targets expected to potentiate the survival and replication of the pathogen in adverse environments. These results may also identify possible candidates useful for the development of vaccines, diagnostic kits or therapies aimed at the reduction of losses in agribusiness. RESULTS: Under the 3 simulated conditions (acid, osmotic and thermal shock stresses), 474 differentially expressed genes exhibiting at least a 2-fold change in expression levels were identified. Important genes to the infection process were induced, such as those involved in virulence, defence against oxidative stress, adhesion and regulation, and many genes encoded hypothetical proteins, indicating that further investigation of the bacterium is necessary. The data will contribute to a better understanding of the biology of C. pseudotuberculosis and to studies investigating strategies to control the disease. CONCLUSIONS: Despite the veterinary importance of C. pseudotuberculosis, the bacterium is poorly characterised; therefore, effective treatments for caseous lymphadenitis have been difficult to establish. Through the use of RNAseq, these results provide a better biological understanding of this bacterium, shed light on the most likely survival mechanisms used by this microorganism in adverse environments and identify candidates that may help reduce or even eradicate the problems caused by this disease.

Draft Genome Sequence of Serratia fonticola LMG 7882T Isolated from Freshwater.

Serratia fonticola is a Gram-negative bacterium with a wide distribution in aquatic environments. On some occasions, it has also been regarded as a significant human pathogen. In this work, we report the first draft genome sequence of an S. fonticola strain (LMG 7882(T)), which was isolated from freshwater.

Draft Genome Sequence of Serratia fonticola UTAD54, a Carbapenem-Resistant Strain Isolated from Drinking Water.

Serratia fonticola UTAD54 is an environmental isolate that is resistant to carbapenems due to the presence of a class A carbapenemase and a metallo-ß-lactamase that are unique to this strain. Its draft genome sequence was obtained to clarify the molecular basis of its carbapenem resistance and identify the genomic context of its carbapenem resistance determinants.

High efficiency application of a mate-paired library from next-generation sequencing to postlight sequencing: Corynebacterium pseudotuberculosis as a case study for microbial de novo genome assembly.

With the advent of high-throughput DNA sequencing platforms, there has been a reduction in the cost and time of sequencing. With these advantages, new challenges have emerged, such as the handling of large amounts of data, quality assessment, and the assembly of short reads. Currently, benchtop high-throughput sequencers enable the genomes of prokaryotic organisms to be sequenced within two hours with a reduction in coverage compared with the SOLiD, Illumina and 454 FLX Titanium platforms, making it necessary to evaluate the efficiency of less expensive benchtop instruments for prokaryotic genomics. In the present work, we evaluate and propose a methodology for the use of the Ion Torrent PGM platform for decoding the gram-positive bacterium Corynebacterium pseudotuberculosis, for which 15 complete genome sequences have already been deposited based on fragment and mate-paired libraries with a 3-kb insert size. Despite the low coverage, a single sequencing run using a mate-paired library generated 39 scaffolds after de novo assembly without data curation. This result is superior to that obtained by sequencing using libraries generated from fragments marketed by the equipment's manufacturer, as well as that observed for mate-pairs sequenced by SOLiD. The generated sequence added an extra 91kb to the genome available at NCBI.

Ion Torrent-based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high-performance liquid chromatography a promising rRNA depletion method.

Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.

The core stimulon of Corynebacterium pseudotuberculosis strain 1002 identified using ab initio methodologies.

Corynebacterium pseudotuberculosis is a bacterium which causes diseases such as caseous lymphadenitis in small ruminants, resulting in large-scale economic losses for agribusiness worldwide. Consequently, this bacterium including its transcriptional profile analysis has been the focus of various studies. Identification of the transcripts that appear under conditions that simulate the environment encountered by this bacterial species in the host is of great importance in discovering new targets for the production of more efficient vaccines. We sequenced the cDNA of Corynebacterium pseudotuberculosis strain 1002, using the SOLiD V3 system, under the following conditions: osmotic stress (2 M), acidity (pH), heat shock (50 °C) and control condition (N). To identify the transcripts shared among the stimulons and integrate this information with the results from BLAST and BLAST2GO, we developed the software CoreStImulon (CSI) which allows the user to individually distinguish the genes in terms of their participation in biological processes, their function and cellular location. In the biosynthetic processes, eleven genes represented in the core stimulon and twenty genes in the control were observed. This validates the hypothesis that the organisms strategy for surviving in a hostile environment is through growth reduction. The oxidation reduction process, response to stress process, and cell adhesion are controlled by genes that contribute to bacterial cell maintenance under stress conditions; these could be involved in their pathogenicity. The methodology for identification of transcripts obtained by ab initio assembly and shared among the stimulons permitted candidates selection for vaccine studies. CSI is available at https://sourceforge.net/projects/corestimulon/.