The whole genome of Staphylococcus aureus MW2, a pathogen causing community-acquired infections, was sequenced and compared with that of the hospital-acquired S. aureus N315 and others to unveil the high toxicity of MW2 infection. Differences in the genomic sequences of N315 and MW2 amount to about 5% and the presence of seven 'genomic islands' was noticed. A new type of island termed 'cassette chromosome' was also recognized in MW2 that leads to the penicillin/cefem-resistance by inserting into the MW2 chromosome. In addition, MW2 was newly found to possess 19 putative virulence factors which may be active and, when brought together, may cause a strong virulence.
This bacterium is one of the major pathogens causing community-acquired infections. In the Midwestern US, several fatal infections caused by MW2 were reported in late 90's. Combination of the distinct allelic forms of genomic islands found in different strains of S. aureus appears to be the genetic basis in determining the pathogenicity of medically important S. aureus strains including MW2. This information may be important for the detection of S. aureus infections in future.
|List of ORFs updated in annotation
We thank Dr. Enrique D. Vines, University of Western Ontario for letting us know about his study.
|Genomic size||2,841,116 bp|
|G+C content||32.80 %|
|Number of ORFs assigned||2,659|
|Percentage of the coding regions||84.57 %|
|Percentage of the intronic regions||0.00 %|
|Number of rRNA genes||
|Number of tRNA genes||
|Number of other features
As in the case of S. aureus N315, the entire genomic nucleotide sequence of S. aureus MW2 genome was determined by sequencing the whole genome shotgun (WGS) clones.
WGS clones were constructed with pUC118 plasmid. For this purpose, genomic DNA fragments of 1.5-3.0 kb in size were generated by random mechanical shearing and the resultant random clones were prepared in Escherichia coli DH5_MCR (Life Technologies, MD, USA). They were then sequenced using the dye-terminator kit and analyzed with an PRISM 3700 DNA analyzer from ABI (CA, USA). Subsequently, 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 were performed using Sequencher (Gene Codes, MI, USA).
The integrity of the assembled data was confirmed by performing PCR amplification of the 18-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 MW2 was resulted from about 64,000 sequences of WGS clones as well as PCR fragments.Criteria for ORF assignment and annotation
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/.