Single-molecule Real-time DNA Research Articles

Adaptive Strategies of Bacillus thuringiensis Isolated from Acid Mine Drainage Site in Sabah, Malaysia

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

Chromosome sorting for the masses?

Chromosome sorting for the masses?

The Progress on Sequencing and Detection of Hydroxymethylated DNA

The recently re-discovered 5-hydroxymethylcytosine (5hmC) is established as the sixth base and found in various mammalian tissues. Although the content of 5hmC is much lower than that of the best known epigenetic mark 5-methylcytosine (5mC), 5hmC plays key roles in nuclear reprogramming, regulates the gene activity, and initiates the DNA demethylation in mammals. The formation of 5hmC results from the oxidation of 5mC in genomic DNA, which is mediated by Tet (ten eleven translocation) family dioxygenases. The Tet-mediated 5mC oxidation has just been identified. The 5hmC formation can cause DNA replication-dependent and passive DNA demethylation, meanwhile, it can also induce active DNA demethylation by further oxidation forming 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be removed by base excision repair. Essentially, the discovery of 5fC and 5caC indicates the occurrence of active DNA demethylation mechanisms. The 5fC and 5caC have been detected in genomic DNA of mouse embryonic stem cells, but 5hmC has been found in various tissues. The 5hmC abundance in genomic DNA of certain tumors is found to be associated with the tumor process. However, the genome-wide distribution and sequence selectivity of 5hmC are not known yet. 5hmC cannot be dis- criminated from 5mC by the well-known bisulfite sequencing. Therefore, it is essential to develop new methods and tech- nologies for detecting and sequencing 5hmC. To this purpose, the two or more of the technologies for modification of 5hmC by glucosylation or chemical labeling, restriction enzymatic digestion, affinity capture, liquid chromatography and mass spectrometry, and bisulfite sequencing are combined. The further combination with next-generation high-throughput DNA sequencing technologies may provide genome-wide information. The single molecule real time DNA sequencing is also of choice to detect 5hmC at the gene level or the genome level. Recently, oxidative bisulfite sequencing was for the first time developed for quantitative mapping of 5hmC in genomic DNA at single base resolution. The genome-wide 5hmC sequencing at single base resolution is also reported. Here we briefly review and discuss the detection and sequencing technologies for 5hmC analysis. Keywords 5-hydroxymethylcytosine; 5-methylcytosine; DNA sequencing