Genomic and taxonomic characterization of strain CCM 2573: Uncovering unique genetic features and description of Macrococcus caseolyticus subsp. lactis subsp. nov.

Strain CCM 2573 is a Gram-positive bacterium that has been intensively studied in the past due to its distinct chemotaxonomic properties, but its reliable taxonomic classification has not been satisfactorily clarified. Whole-genome sequencing and comparative genomic analyses performed in this study revealed that the strain belongs to the Macrococcus caseolyticus phylogenetic clade. Genome-to-genome comparisons confirmed the closest relationship to the type strains of M. caseolyticus subsp. hominis CCM 7927^T and M. caseolyticus subsp. caseolyticus DSM 20597^T. However, the strain harbored unique genomic elements distinguishing it from its nearest phylogenetic neighbors. Its accessory genome contains dozens of insertion sequences, a 92-kbp composite transposon with unique palindromic repeat loci associated with a CRISPR-Cas adaptive immune system, a pseudo-staphylococcal chromosome cassette, and several additional genomic islets. Unlike other macrococci, strain CCM 2573 exhibits a specific peptidoglycan composition (L-Lys-Gly₂-Ser₂-Gly) and shows a higher phylogenetic divergence of aminoacyltransferases (FemABX) involved in interpeptide bridge synthesis. In addition, it reveals distinct biochemical characteristics from both subspecies of M. caseolyticus, particularly in its ability to produce acid from galactose, cellobiose, melezitose, and turanose, as well as in its susceptibility to novobiocin. The MALDI-TOF mass spectra enable differentiation of the strain from other type strains of the genus Macrococcus. The results of polyphasic taxonomy obtained in this study showed that strain CCM 2573 belongs to the species M. caseolyticus, but it is distinct from both validly named M. caseolyticus subspecies. We propose to assign the analyzed strain as a new subspecies, Macrococcus caseolyticus subsp. lactis subsp. nov. The type strain is CCM 2573^T (= DSM 20227^T).