Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus

Abstract

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.

The Cell Wall Stress Stimulon of Staphylococcus aureus and Other Gram- Positive Bacteria

“…fmtA was discovered to be the interrupted gene in a Tn551 insertion mutant of MRSA COL that became more susceptible to oxacillin in the presence of 0.02% Triton X-100 [59]. Further investigation revealed FmtA to be a membrane-associated protein that lacks penicillin-binding activity even though it has two of three conserved motifs typical of PBPs and β-lactamases [60].…”

Resistant penicillin-binding proteins

“…Mutants in femD and femF are defective in cytoplasmic steps of peptidoglycan precursor synthesis [78,79]. Other genes such as llm [80] and fmt [81] can affect methicillin resistance without changing PBP2% production. The impact of these different factors on methicillin resistance in clinical isolates has not yet been measured.…”

Variation in synergistic activity by flavone and its related compounds on the increased susceptibility of various strains of methicillin-resistant Staphylococcus aureus to β-lactam antibiotics

“…With respect to the susceptibility of MRSA to ␤-lactams, several compounds, such as baicalin [11], diterpenes [12], epigallocatechin gallate [13,14], epicatechin gallate [15], flavone and its derivatives [16,17], polidocanol [18], polyoxtungstates [19,20], tellimagrandin I [21], triazine dye [22], tripeptide [23] and Triton X100 [24][25][26][27][28], have only a weak antibacterial effect against MRSA, but synergistically decrease the MIC of ␤-lactams of MRSAs. However, there is no clear explanation for this interesting phenomenon of synergism.…”