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:After the wild type strain of Vibrio natriegens ATCC 14048 (BioSample Nr. NCBI: SAMN03178087) was cured from prophages (as described in the material and methods section), the genomes of the resulted strains dvnp1, dvnp2 and vnp12 were sequenced. As a control, the genome of the wild type strain was also prepared and used for sequencing. The aim of the sequencing was to verify the deleted regions and to screen the genome for new mutations.
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.
Project description:Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated by metabolic enrichment on levoglucosan as sole carbon source. Genome sequencing and proteomics analysis revealed expression of a series of gene clusters encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan b-eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident. The expressed gene clusters contained a range of putative sugar phosphate isomerase/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbors revealed that homologues of LgdA, LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One sugar phosphate isomerase/xylose isomerase cluster (LgdB3) was identified with limited distribution mutually exclusive with LgdB2. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight the diversity within the LGDH pathway through which bacteria utilize levoglucosan as a nutrient source.
Project description:Although many members of the genus Vibrio are known to inhabit the marine photic zone, an understanding of the influence of light on the molecular physiology of Vibrio spp. has largely been neglected. To begin to characterize the photophysiology of one such Vibrio sp. (Vibrio campbellii ATCC strain BAA-1116) we used microarray-based expression profiling to compare the transcriptomes of illuminated versus dark cell cultures. Specficially, we compared the transcriptomes of wild type V. campbellii (STR) cells that were cultured in M9 minimal salts medium plus glucose under two conditions: (i) after 24 hours of continuous dark and (ii) after a 12 hour dark:12 hour light cycle (white light illumination at 54 µmol photons s-1 m-2). The results revealed a large photostimulon (differential expression of ~20% of the V. campbellii genome; adjusted p value < 0.0001) that surprisingly included ~75% of the type III secretion system (T3SS) genes which were found to be 1.6 – 5.4X more abundant in illuminated cultures. These findings, which were confirmed by quantitative reverse transcription PCR and quantitative membrane proteomics, strongly suggest that the photostimulon of strain BAA-1116 includes the T3SS.