Project description:Purpose:The goals of this study are to clarify the B. subtilis NBRC 16449 response to soybeans. Methods: B. subtilis NBRC 16449 cells were aerobically cultured in liquid LB, LB solidified with agar, or on surface of boiled soybeans to logarithmic growth phase. Total RNAs were extracted from bacterial cells by Hot-Phenol method. Samples for RNA-seq were prepared according to Illmina protocol available from the manufacture. The sequence reads that passed quality filters were analyzed at the transcript isoform level with bowtie v0.11.2. Results: Using an optimized data analysis workflow, we mapped around 15 million sequence reads per sample to the whole genome of B. subtilis BEST195 and identified 4271 transcripts in B. subtilis NBRC 16449 with Bowtie aligner. Read count per genome was extracted from known gene annotations with HTSeq program. Compared the transcriptomes of B. subtilis NBRC 16449 grown on LB solidified with agar to that grown on surface of boiled soybeans, about 5% of genes showed the different expression levels.
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:Purpose: We investigate the evolutionary footprints of a bacteria-plasmid association consisting of Escherichia coli K-12 MG1655 and plasmid RP4 undergoing a long-term sub-MIC antibiotic stress. Methods: Bacterial mRNA profiles of evolved RP4-carrying strains (E:H:p) and ancestral RP4-carrying strains (A:H:p) were generated by deep sequencing on an Illumina Hiseq platform. The sequence reads that passed quality filters were analyzed by Burrows–Wheeler Aligner (BWA), followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads of E:H:p and 12 million sequence reads of A:H:p to the E. coli MG1655 genome (GCF_000801205.1) and differential expressed genes were identified with TopHat workflow. RNA-seq data showed that approximately 15% of the transcripts showed differential expression between the E:H:p and A:H:p strains, with a fold change ≥1 and p value <0.005. Altered expression of 26 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Data analysis with bowtie and TopHat workflows provided complementary insights in transcriptome profiling. Conclusions: Our study showed the coevolved bacteria-plasmid pairs has colonization traits superior to the wild-type parent strain. Antibiotic stress was necessary for bacterial evolution and evolved strains mostly employed transcriptional modifications to reduce plasmid-related cost in evolutionary adaptations. Several genes related to chromosome-encoded efflux pumps were transcriptionally upregulated, while most plasmid-harboring genes were downregulated based on RNA gene sequencing. These transcriptional modifications endowed evolved strains with resistant phenotype modifications, including the enhanced bacterial growth and biofilm formation.