FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: Molecular cloning and characterization

Abstract

The methicillin resistance determinant (mec) in Staphylococcus aureus resides on additional DNA not present in isogenic sensitive cells. However, besides mec, other chromosomally determined factors are essential for expression of methicillin resistance. We cloned and characterized a chromosomally determined gene which encodes a factor essential for the expression of methicillin resistance (femA) in S. aureus. femA mapped in chromosomal segment number 18, genetically very distant from the methicillin resistance determinant (mec). The product of femA was a protein of an apparent size of 48 kDa. FemA restored methicillin resistance in S. aureus that had become sensitive to methicillin by insertion of omega 2003 (femA::Tn551). Although FemA was needed for cell growth in the presence of beta-lactam antibiotics, it had no influence on the synthesis of the low affinity, additional penicillin-binding protein (PBP2') encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin. Nucleotide sequence analysis, Northern RNA blotting and S1 nuclease RNA mapping suggested that femA was transcribed on a polycistronic mRNA. This mRNA contained the coding region (ORF419) producing a protein of 47 kDa. The nucleotide and amino acid sequence of FemA showed homologies with ORF419, suggesting that these genes arose by gene duplication. In addition we present evidence for a second chromosomal factor, femB, involved in expression of methicillin resistance which maps close to femA.

Kinetic Characterization of Lipid II-Ala:Alanyl-tRNA Ligase (MurN) from Streptococcus pneumoniae using Semisynthetic Aminoacyl-lipid II Substrates

“…The MurN gene product shares 26% sequence identity with Staphylococcus aureus FemA, which catalyzes the addition of the second and third glycine residues in the branched muropeptide of S. aureus (38,39). Disruption of the femA gene abolishes methicillin resistance in this organism (40). The Fem-ABX proteins have a requirement for aminoacyl-charged tRNAs as substrates for the nonribosomal peptide bond formation of the pentaglycine bridge (40).…”

“…Disruption of the femA gene abolishes methicillin resistance in this organism (40). The Fem-ABX proteins have a requirement for aminoacyl-charged tRNAs as substrates for the nonribosomal peptide bond formation of the pentaglycine bridge (40). The functional and sequence similarity of MurN to these proteins suggested that it also uses aminoacyl-tRNAs as substrates.…”

Detection ofmecA, femA, andfemBgenes in clinical strains of staphylococci using polymerase chain reaction

“…Based on the nucleotide sequences of mecA, femA and femB genes [4,16], the oligonucleotides listed in Table 1 Figure 1 shows electrophoretic patterns of the DNA products after PCR using three representative oxacillin-RSA strains. The DNA fragments of 533, 509 and 651 bp were amplified from mecA, femA, and femB genes, respectively.…”

“…Besides the mec regulator genes, femA and femB genes on the chromosome have been shown to encode proteins which considerably affect the level of methicillin resistance of S. aureus [16]. Although fem genes were suggested to be specific for S. aureus [17], distribution of these genes in staphylococci has not been fully established.…”

“…The first aim of this study was to develop a system that differentiated MRSA and mecA-positive CNS by the detection of fem genes together with the mecA gene by PCR. Secondly, to determine whether or not the presence offem and mecA genes in clinical isolates were associated with a high level of drug resistance as observed for certain S. aureus strains [16]. MATERIALS [18], such strains were described as oxacillin-RSA (oxacillin-resistant S. aureu-s) in this study in order to distinguish it from the term 'MRSA' which indicated S. aureus that possessed the mecA gene.…”