Project description:Leishmania donovani WHO reference strain MHOM/IN/80/DD8 and Leptomonas seymouri isolates Ld 2001 and Ld39 were used for proteome analysis which were originally isolated from clinical cases of kala azar patients with different inherent antimonial sensitivities. Ld 2001 was Sb-S and Ld 39 was Sb-R. The genome sequencing of these isolates had confirmed co-infection with Leptomonas.
Project description:Most proteogenomic approaches for mapping single amino acid polymorphisms (SAPs) require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. We present a new strategy for direct SAP detection without relying on NGS data. Among the 348 putative SAP peptides identified in an industrial yeast strain, 85.6% of SAP sites were validated by genomic sequencing.
Project description:Gene content comparison of control C.j. strain 11168 which colonizes and causes disease in a murine model versus strain NW which colonizes but does not elicit disease symptomology in the mouse model. Keywords: DNA/DNA comparison Overall design: Two genome comparison of disease strain versus non disease strain of C.j., Biological replicates - 2, independently grown and harvested, Technical replicates 2, also independent, 3 ORF replicates per array.
Project description:Whole transcriptome analysis of N. gonorrhoeae FA19 and isogenic NGEG_00293 (misR) mutant using RNA-Seq (note that NGO0177 is the misR ORF designation in mapping strain FA1090) Examination of total transcriptomes in N. gonorrhoeae FA19 WT and an FA19 misR::kan isogenic mutant to determine the regulatory impact of the MisR response regulator on cells grown under laboratory conditions. Illumina HiSeq-2000 next generation sequencing was used to sequence the transcriptomes of each strain. Reads were mapped against the N. gonorrhoeae FA1090 genome (NCBI accession number NC_002946) because at the time this experiment was run the FA19 genome was incomplete.
Project description:The most important approach to the development of platform organisms for recombinant protein production relies on random mutagenesis and phenotypic selection. Complex phenotypes, including those associated with significant elevated expression and secretion of heterologous proteins, are the result of multiple genomic mutations. Using next generation sequencing, a parent and derivative hypersecreter strain (B41) of Escherichia coli were sequenced with an average coverage of 52.8X and 55X, respectively. A new base-pair calling program, revealed a single nucleotide polymorphism in the B41 genome at position 1,074,787, resulting in translation termination near the N-terminus of a transcriptional regulator protein, RutR, coded by the ycdC gene. We verified the hypersecretion phenotype in a ycdC::Tn5 mutant and observed a 3.4-fold increase in active hemolysin secretion, consistent with the increase observed in B41. mRNA expression profiling showed decreased expression of tRNA-synthetases and some amino acid transporters in the ycdC::Tn5 mutant. This study demonstrates that power of next generation sequencing to characterize mutants leading to successful metabolic engineering strategies for strain improvement. Overall design: 6 samples were analyzed with three biological replicates for each strain. Hly-ycdC+ is the control strain.
Project description:We investigated the gene expression and metabolic regulatory mechanisms associated with the high-level accumulation of ICPs by performing the transcriptomics analysis of B. thuringiensis strain CT-43, using Illumina high throughout sequencing (RNA-seq) technique. The bacterial cells were collected at the time points of 7 h, 9 h, 13 h and 22 h for the whole-genome transcriptomics, respectively.
Project description:Industrial production of penicillins with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. Sequencing of the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 revealed many genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid, the amino-acid precursors for penicillin biosynthesis, as well as genes encoding microbody proteins, increased in the high-producing strain. Many key (intra)cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven functional analysis. Keywords: genetic modification Overall design: Transcriptome analysis was performed on aerobic, glucose-limited chemostat cultures (D=0.03h-1)of the Wisconsin54-1255 strain and the derived industrial, penicillinG high-producing strain DS1769032. Chemostat cultivation offers the possibility to keep specific growth rate and other key parameters constant33. Some intermediates of β-lactam biosynthesis are produced in the absence of the side-chain precursor phenylacetic acid (PAA), but penicillinG biosynthesis is strictly dependent on PAA addition to culture media.
Project description:The aims of this study were to present modifications to the annotations of the genome of C. posadasii, one of two closely related species of Coccidioides, a dimorphic fungal pathogen that causes coccidioidomycosis, also called Valley Fever. Proteins present in lysates and filtrates of in vitro grown mycelia and parasitic phase spherules from C. posadasii strain Silveira were analyzed using a GeLC-MS/MS method.
Project description:Clavibacter michiganensis subsp. michiganensis is an important Gram-positive phytopathogenic bacteria that causes bacterial wilt and canker in tomato. The genome of the type strain, NCPPB382, has been sequenced and annotated, however comparative genomics suggests that certain regions are under- or misannotated. In order to improve the genome annotation, we have undertaken a proteogenomic study of this important pathogen. Samples were grown in culture and the proteome of the pellet and supernatant were analyzed separately using shotgun HPLC-MS/MS. These proteomics datasets were analyzed and a number of missing gene were found and a number of existing gene calls were modified.
Project description:P. multocida is the causative agent of a wide range of diseases of animals, including fowl cholera in birds. Fowl cholera isolates of P. multocida generally express a capsular polysaccharide composed of hyaluronic acid. There have been reports of spontaneous capsule loss in P. multocida fowl cholera-causing strains but the mechanism by which this occurs has not been determined. In this study, we identified three independent strains that had spontaneously lost the ability to produce capsular polysaccharide. Quantitative RT-PCR showed that these strains had significantly reduced transcription of the capsule biosynthetic genes, but DNA sequence analysis identified no mutations within the cap biosynthetic locus. However, whole genome sequencing of paired capsulated and acapsular strains identified a single nucleotide polymorphism within fis that was present only in the acapsular strain. Sequencing of fis from two independently derived spontaneous acapsular strains showed that each contained a mutation within fis. Complementation of these strains with an intact copy of fis returned normal capsule expression to all strains. Therefore, expression of a functional Fis protein is absolutely required for normal capsule expression in P. multocida.DNA microarray studies comparing one of the acapsular pairs (AL114 to AL1115) identified approximately 30 genes as down-regulated in the mutant; including pfh_B2 which encodes the filamentous hemagglutinin, a known P. multocida virulence factor and the cross protective surface antigen plpE. Overall design: Biological triplicates of each strain were analysed in a single colour experimental design