Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.
The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.
Vannuffel
P
P
Laboratory of Applied Molecular Technology, Brussels, Belgium. vannuffel@ebcm.ucl.ac.be
Heusterspreute
M
M
Bouyer
M
M
Vandercam
B
B
Philippe
M
M
Gala
J L
JL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D016384:Consensus Sequence; D004271:DNA, Fungal / Q000737:chemistry; D008969:Molecular Sequence Data; D009716:Numerical Analysis, Computer-Assisted; D016415:Sequence Alignment; D013045:Species Specificity; D013210:Staphylococcus / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004271:DNA, Fungal; C096686:FemA protein, Bacteria