Staphylococcal cassette chromosome mec-like element in Macrococcus caseolyticus

Abstract

Macrococcus is a bacterial genus that is closely related to Staphylococcus, which typically is isolated from animal skin and products. The genome analysis of multidrug-resistant Macrococcus caseolyticus strain JCSC5402, isolated from chicken, previously led to the identification of plasmid pMCCL2, which carries a transposon containing an unusual form of the Macrococcus mec gene complex (mecA(m)-mecR1(m)-mecI(m)-blaZ(m)). In M. caseolyticus strain JCSC7096, this mec transposon containing the mec gene complex (designated Tn6045 in this study) was found integrated downstream of orfX on the chromosome. Tn6045 of JCSC7096 was bracketed by the direct repeat sequences (DR) specifically recognized by cassette chromosome recombinase (CCR). A non-mecA-containing staphylococcal cassette chromosome (SCC) element, designated SCC(7096), was integrated next to the mec transposon and separated from the latter by a DR. Nested PCR experiments showed that the mec transposon not only was excised singly but also coexcised with SCC(7096) from the chromosome at the DRs. The coexcised elements formed the extrachromosomal closed circular DNA of the SCCmec-like element. SCCmec is known to be the mobile element conveying methicillin (meticillin) resistance in staphylococci. However, its origin has been unknown. Our observation revealed a potential mechanism of the generation of a new SCCmec-like element in M. caseolyticus, a commensal bacterium of food animals.

FIG. 1.

Localization of mecA_m by S1-PFGE-based Southern hybridization. (a) The S1-PFGE analysis indicated the existence of a large plasmid. Agarose gel plugs of total cellular DNA were digested with S1 nuclease, and the resulting fragments were separated on an agarose gel and stained with ethidium bromide. Lane 1, molecular size marker; lane 2, JCSC5402; lane 3, JCSC7096; lane 4, JCSC7528. The white arrows indicate the sizes of plasmids. (b) Southern hybridization-localized mecA_m gene. Lane numbers in panel b correspond to those in panel a. The black arrows indicate the size of the DNA fragment carrying the mecA_m gene.

FIG. 2.

Genome analysis of Macrococcus caseolyticus. (a) Structure of the downstream of orfX in the Macrococcus caseolyticus chromosome and comparison to that of the MRSA N315 SCCmec. DRs are shown by arrowheads. The conserved DNA regions are shown by the light color between the corresponding regions of two chromosomes. mecI and mecR1 encode repressors and signal transducers for mecA gene expression, respectively. mecA encodes PBP2′. blaZ encodes beta-lactamase. ccrA and ccrB encode site-specific recombinases. IS431 is an insertion sequence identified upstream of the mecA gene among all the extant SCCmec elements found in staphylococcal species. The psm-mec gene is identified only in the downstream of the mecI gene in the type II and III SCCmec. Arrows indicate the ORFs and their directions of transcription. (b) The excision sites for the spontaneous loss of the mec transposon Tn6045 and ΨSCCmec₇₀₉₆, SCC₇₀₉₆, and ΨSCC_7096-2 from the JCSC7096 chromosome. The orfX gene, ΨSCCmec₇₀₉₆ containing Tn6045, SCC₇₀₉₆, and ΨSCC_7096-2 are represented by purple, light green, pink, and light blue bars, respectively. Two DRs (DRtnL and DRtnR) were involved with the singular excision of the mec transposon. DR1 to DR4 were involved with the spontaneous loss of the SCC or ΨSCC element together with the mec transposon. IRs are indicated by the underline. Arrowheads indicate the location and directions of primers used for the nested PCR assay.

FIG. 3.

Phylogenetic analysis. (a) Phylogenetic trees of ccr genes. The two site-specific recombinases are the integrase (int) of bacteriophage ΦFC1, found in Enterococcus faecalis, and the site-specific recombinase found in Clostridium acetobutylicum ATCC 824. The ccrA and ccrB genes used were the following: ccrA_m1 and ccrB_m1 from M. caseolyticus strain JCSC7096; ccrA1 and ccrB1 from S. aureus strain MSSA476; ccrA2 and ccrB2 from MRSA strain N315; ccrA3 and ccrB3 from MRSA strain 85/2082; and ccrA4 and ccrB4 from MRSA strain CHE482. (b) The phylogenetic tree of mecA and pbp genes. The BLASTX was performed with M. caseolyticus JCSC5402 pbp1 to pbp4 and mecA_m nucleotide sequences as the query. The phylogenic trees were drawn based on the mutual degrees of homology of the hit sequences. S. aureus pbp1 to pbp4 are from MRSA strain MW2, S. epidermidis pbp1 to pbp3 are from strain RP62A, S. haemolyticus pbp1 to pbp4 are from strain JCSC1435, S. saprophyticus pbp1 to pbp4 are from strain ATCC15305, M. caseolyticus pbp1 to pbp4 are from strain JCSC5402, E. hirae pbp5 is from strain R40, E. faecium pbp5 is from strain D63R, E. hirae pbp3r is strain S185R, E. faecalis pbp4 is from strain V583, S. aureus mecA is from strain N315, S. vitulinus mecA is from strain CSWY15, and S. sciuri mecA is from strain ATCC 29062.