Project description:A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium isolates obtained from a variety of host species. DNA microarrays were used as a platform for comparing mycobacteria field isolates with the sequenced bovine isolate Mycobacterium avium subsp. paratuberculosis (Map) K10. ORFs were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to Map K10 DNA. Map isolates cultured from cattle, bison, sheep, goat, avian, and human sources were hybridized to the Map microarray. Three large deletions were observed in the genomes of four Map isolates obtained from sheep and four clusters of ORFs homologous to sequences in the Mycobacterium avium subsp. avium (Maa) 104 genome were identified as being present in these isolates. One of these clusters encodes glycopeptidolipid biosynthesis enzymes. One of the Map sheep isolates had a genome profile similar to a group of Mycobacterium avium subsp. silvaticum (Mas) isolates which included four independent laboratory stocks of the organism traditionally identified as Maa strain 18. Genome diversity in Map appears to be mostly restricted to large sequence polymorphisms that are often associated with mobile genetic elements. Keywords: Comparative genomic hybridization
Project description:Mycobacterium avium is one of the prominent disease causing bacteria in humans. It causes lymphadenitis, chronic pulmonary and extrapulmonary and disseminated infections in adults, children and immunocompromised humans. M. avium has ~4,500 predicted gene models, out of which not all are identified at proteomic level. Proteomic database search followed by proteogenomic analysis helps in the correction of gene models, identification of novel exons/genes, variant proteins. As part of this study, we performed proteomic analysis of M. avium cultures by data-dependent acquisition (DDA) and data-independent acquisition (DIA) method followed by proteogenomic analysis of M. avium proteomic data. M. avium culture was subjected to proteomic sample preparation. The resulting peptides were acquired in 120min DDA (12 bRPLC) and DIA method using EASY nLC 1200 liquid chromatogram system coupled to Orbitrap Fusion Tribrid mass spectrometer. The resulting DDA raw files were searched sequentially against the M. avium proteome database, Mycobacterium tuberculosis H37Rv and Ra proteome database (Mtb), M. avium genome six-frame translated proteome and variant proteins database, respectively. The database search result was converted into a spectral library and used for DIA data search for the validation of GSSPs and variant peptides. The database search of M. avium DDA has resulted in the identification of 2,954 M. avium proteins, 128 Mtb proteins, 174 GSSPs (M. avium genome six-frame translated proteome) and 795 SNPs corresponding to 612 proteins (variant proteins database), respectively. The M. avium proteome database search has covered 62.09% of the proteome with the identification of 2,954 proteins out of 4,757 proteins in the database. From the manual categorization of 174 GSSPs, we observed 23 N-terminal extensions, 142 pseudogene coding peptides and one each novel exon and short peptide, respectively.
Project description:We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate genome-wide temporal chromatin maps of Chlamydia trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal regions of chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work will serve as a basis for future functional studies of transcriptional regulation and epigenomic regulatory elements in Chlamydia-infected human cells.
Project description:Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium TMC724. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these coincided with the start codons and therefore belong to leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 9 intergenic small RNAs were mapped. Four of the revealed intergenic small RNAs, including igMAV_1034-1035 expressed at a very high level, have no homologs in M. tuberculosis, whilst M. avium lacks several intergenic sRNAs present in M. tuberculosis. Among those, MTS479 and MTS1338 are of special interest due to their possible implication in pathogenesis. Elucidation of differences in the repertoire of intergenic sRNAs between the two mycobacterial species may improve our understanding of mycobacterial diseases pathogenesis. Transcriptional profile of Mycobacterium avium TMC724, grown at 37M-BM-0C in Dubos broth until mid-logarithmic growth phase