Complete genome sequence of Macrococcus caseolyticus strain JCSCS5402, [corrected] reflecting the ancestral genome of the human-pathogenic staphylococci

Abstract

We isolated the methicillin-resistant Macrococcus caseolyticus strain JCSC5402 from animal meat in a supermarket and determined its whole-genome nucleotide sequence. This is the first report on the genome analysis of a macrococcal species that is evolutionarily closely related to the human pathogens Staphylococcus aureus and Bacillus anthracis. The essential biological pathways of M. caseolyticus are similar to those of staphylococci. However, the species has a small chromosome (2.1 MB) and lacks many sugar and amino acid metabolism pathways and a plethora of virulence genes that are present in S. aureus. On the other hand, M. caseolyticus possesses a series of oxidative phosphorylation machineries that are closely related to those in the family Bacillaceae. We also discovered a probable primordial form of a Macrococcus methicillin resistance gene complex, mecIRAm, on one of the eight plasmids harbored by the M. caseolyticus strain. This is the first finding of a plasmid-encoding methicillin resistance gene. Macrococcus is considered to reflect the genome of ancestral bacteria before the speciation of staphylococcal species and may be closely associated with the origin of the methicillin resistance gene complex of the notorious human pathogen methicillin-resistant S. aureus.

FIG. 1.

Circular representation of the chromosome of M. caseolyticus JCSC5402. The first (outermost) circle shows the nucleotide position in base pairs. Red arcs indicate prophages, whereas yellow shows the insertion sequences. The second circle shows ORFs oriented in the forward direction, whereas the third circle indicates those oriented in the reverse direction. Green bars in the second and the third circles represent unique ORFs of M. caseolyticus, whereas blue bars show the ORFs that are the most similar to staphylococcal genes. Orange bars indicate the first-hit Bacillus ORFs, and red bars indicate those whose homologs are not found in staphylococcal genomes and yet first-hit Bacillus ORFs. Black bars indicate ORFs whose most homologous genes were not found in either Bacillus or Staphylococcus. The fourth and fifth circles show genes for transfer RNAs and rRNAs, respectively. The sixth circle represents the regions with high GC content (≥50%; purple) and low GC content (blue). The window size and shift increment for GC content were 5 kbp and 0.1 kb, respectively. The seventh circle shows the G-C skew with positive (yellow) and negative (blue) values.

FIG. 2.

Genome alignment between M. caseolyticus JCSC5402 and S. aureus MW2 (top) or between M. caseolyticus JCSC5402 and B. subtilis 168 (bottom). Red dots indicate homologous regions that were drawn by using In Silico molecular cloning software. The top panel includes genomic island locations and their names in either purple (unique to M. caseolyticus JCSC5402, vertical scripts) or sky blue (unique to S. aureus, horizontal scripts). SCCmec is a determinant for methicillin resistance (17). The νSaα and νSaβ are pathogenicity islands present in all of the S. aureus genomes thus far sequenced, whereas the νSa3 element is found in some S. aureus strains, including MW2 (2, 3). φSa elements represent prophages.

FIG. 3.

Tandem location of beta-lactam resistant genes mecA (designated mecA_m) and blaZ in M. caseolyticus JCSC5402/pMCCL2 compared to mec and bla complexes in S. aureus N315. The numbers in the purple shaded areas represent mutual amino acid sequence identities.

FIG. 4.

Distribution of conserved ortholog groups among Bacillaceae and Staphylococcaceae families and Listeria innocua. After identifying common orthologs among various species, we constructed the conserved chromosomal structure (“core structure”) on the basis of the consensus arrangement of the conserved orthologs. An ortholog group in the resulting core structure is indicated as a colored line across horizontal black line representing a chromosome. To simplify the figure, only universally conserved, one-to-one correspondence ortholog groups are shown. To visualize chromosomal rearrangement of the core structure, color gradation is assigned according to the location on the M. caseolyticus JCSC5402 chromosome from red to yellow to green. The replication origins (oriC) are located at the center.

FIG. 5.

Phylogenetic relationships among gram-positive bacteria. The tree diagram was generated by using the concatenated protein sequence of 297 orthologous core genes that are conserved in all of the tested genomes in one-to-one correspondence. These genes are shown as colored lines in Fig. 4.