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:We quantified bacterial incorporation of algal-derived complex dissolved organic C (DOC) and N (DON) and net algal incorporation of remineralized C and N at the single cell level using isotope tracing and NanoSIMS for fifteen bacterial co-cultures growing with the diatom Phaeodactylum tricornutum, and examined the expressed proteins of two of the isolates when growing with P. tricornutum. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline.
Project description:Phytoplankton blooms represent hotspots of primary production and lead to the formation of particulate organic matter composed of living and dead algal cells. These particles are characterized by steep chemical gradients, for instance in oxygen concentration, that provide diverse ecological niches for specifically adapted microbes to thrive. Particulate fractions were collected at almost daily intervals between early March and late May in 2018. Amplicon sequencing and Meta-omics was used to asses microbial community composition and functionality at different time points.
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
Project description:Given the overwhelming evidence that symbiont genotypes differentially affect host processes such as growth, bleaching susceptibility, and nutrient acquisition, we set out to measure gene expression differences in fragments of Montastraea faveolata harboring two different clades of Symbiodinium. On the reefs near Puerto Morelos, México, colonies of M. faveolata are known to shift algal symbiont clade with depth, often associating with clade A at the top, clade B in the middle, and clade C near the bottom of the colony. By measuring photosynthetic efficiency and gene expression in control and heat-stressed fragments containing either clade B, clade C, or a mix of both, we found that: 1) the algal response to thermal stress is due to both host and algal factors; 2) fragments of M. faveolata express different genes in response to sub-bleaching thermal stress depending on algal genotype; 3) the overall effect of heat stress on coral gene expression is less significant than the effect of housing different zooxanthellae types. Overall, we present convincing evidence that different Symbiodinium clades may be functionally distinct, which in turn, greatly influences host gene expression.