Many bacteria belonging to the genus Staphylococcus are pathogenic to human, of which S. aureus is the most virulent. Other staphylococcal species are less virulent, but they also cause hospital infections especially in immuno-compromised patients. S. haemolyticus is known to colonize on the skin of virtually all human individuals and can be commonly isolated from axillae, perineum, and inguinal areas and is notorious for its multi-drug resistance. Indeed, it was this bacterium that was reported to have acquired resistance to methicillin and glycopeptide antibiotics such as Teicoplanin and Vancomycin in historically early stages. Also, S. haemolyticus can frequently be isolated from human blood cultures and is suspected to cause septicemia, peritonitis, otitis, and urinary tract infections.
To elucidate the mechanism responsible for the apparent genetic plasticity of the genome of S. haemolyticus that might result in its multi-drug-resistance phenotype and to understand its evolutionary relationship with other staphylococcal species, in particular the two S. aureus species whose genomes were previously analyzed at NITE, whole genome sequencing of S. haemolyticus strain JCSC1435 was initiated. The genome is 2.69 Mb in size with the G+C content of 32.8 % and shows about 40 % overall similarity to the genomes of S. aureus species. It contains three small plasmids. One of the most striking features of the genome of this bacterium is its richness in repeat sequences including a large number and variety of insertion sequences (IS's) as well as other types of repeats. The length of these repeats totals as much as some 5 % of the genome. It is suspected that some of the IS's thus identified might have been involved in the transfer of antibiotic resistance to other related staphylococcal species.
|Hundreds of microbial genomes have already been completely sequenced including those analyzed at NITE. To further enhance the usefulness of the results of genome analysis, we added a new feature to the DOGAN to present comparative genomics data. We analyzed the relationships among all genes/ORFs in the five staphylococcal genomes and the results were incorporated into the DOGAN. You can compare genes/ORFs in the genomes and get the syntenic regions or points of possible genomic rearrangements displayed in the dotplot viewer.|
|Genomic size||2,697,861 bp|
|G+C content||32.78 %|
|Number of ORFs assigned||2,694|
|Percentage of the coding regions||86.84 %|
|Percentage of the intronic regions||0.00 %|
|Number of rRNA genes||
|Number of tRNA genes||
|Number of other features
Genome analysis of S. haemolyticus strain JCSC1435 was performed as described below, which was carried out in collaboration with Prof. K. Hiramatsu and his colleagues at the Juntendo University.
The entire genomic nucleotide sequence of S. haemolyticus strain JCSC1435 was established by sequencing whole genome shotgun (WGS) clones. To prepare WGS clones, cells of S. haemolyticus strain JCSC1435 were disrupted by sonification and the genomic DNA obtained was sheared by using a Hydroshear (Gene Machines, San Carlos, California, USA). The genomic fragments of 1.3-2.5 kb in size were subsequently inserted into the Sma I site of pUC18 and the resultant clones were propagated in Escherichia coli K-12 strain DH5αMCR (GIBCO-BRL, Gaithersburg, Maryland, USA). Templates for nucleotide sequencing were prepared by PCR amplification with LA Taq (Takara, Kyoto, Japan) of the DNA directly prepared from colonies. They were subjected to sequencing from both ends with Big-dye Terminator FS Ready Reaction kit followed by analysis on PRISM 3700 DNA Analyzers of Applied Biosystems (Foster City, California, USA) or on Base Station DNA Fragment Analyzers of MJ Research (Waltham, Massachusetts, USA). A total of some 86,000 sequence reads were thus obtained.
The collected data were assembled by using Phred/Phrap. Gaps remaining between resultant contigs were closed by PCR amplification of the corresponding genomic regions by using primers placed at the ends of the flanking contigs followed by sequencing of the PCR products. Editing of the assembled data of the chromosomal and plasmid sequences was performed using Sequencher (Gene Codes, MI, USA). The integrity of the assembled data was confirmed by performing PCR amplification of the 10-15-kb-long genomic regions by generating primers based on the sequence data followed by restriction enzyme cleavage pattern analysis. Also, pulsed-field gel electrophoretic patterns of the genomic fragments with 8 base cutter restriction enzymes were compared with those deduced from the sequence data. Thus, the final genomic sequence of S. haemolyticus strain JCSC1435 was resulted from about 86,000 sequences of WGS clones as well as PCR fragments.
Initial identification of ORFs consisting of 30 or more codons was achieved by using the Glimmer and RBSfinder software and the predicted ORFs were examined individually with the Gambler software. Mutually overlapping ORFs were manually examined one after another. Homology of the proteins encoded by individual ORFs was then searched for against a non-redundant protein database using Blast. The transfer RNA and tmRNA genes were identified, respectively, by the tRNAscan-SE software or by the procedure described at http://www.indiana.edu/~tmrna/.