Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus

Abstract

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 pug/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 jig/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants.Insertion of Tn.918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.Methicillin-resistant strains of Staphylococcus aureus produced a low-affinity penicillin-binding protein (PBP), PBP2' or PBP2a, in addition to the ordinary PBPs (16, 39). PBP2' was considered to take over the function of peptidoglycan biosynthesis from ordinary PBPs in the presence of otherwise inhibitory concentrations of P-lactam antibiotics such as methicillin because of the low affinity of PBP2' for r-lactam antibiotics (11,32). While the production of PBP2' was known to be prerequisite for methicillin resistance, earlier reports indicated that the amount of PBP2' produced did not correspond to the resistance level; that is, strains with a high level of resistance did not necessarily produce a larger amount of PBP2' than strains with a low level of resistance (4, 17, 28). These observations were subsequently confirmed and extended by the finding that mecA, the structural gene for PBP2', was not the single factor determining the resistance level-another genetic determinant(s) was involved in the expression of the high-level methicillin resistance, thus differentiating high-level resistant strains from low-level ones (29). Furthermore, transposon insertional mutagenesis with Tn551 revealed that, for the expression of both low-level and high-level methicillin resistance, several other genetic determinants such as fem genes or auxiliary genes w...

Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis

“…In the absence of WTA, Atl binding on the cell surface is delocalized, causing lysis (21). Furthermore, WTA are required for ␤-lactam resistance in methicillin-resistant S. aureus, and the cells which lack WTA are sensitized to ␤-lactam-induced cell lysis (26)(27)(28). The genes involved in teichoic acid biosynthesis are called the tar genes (for teichoic acid ribitol).…”

Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus

“…During the last 60 years S. aureus increasingly acquired resistance genes to counteract extensive antibiotic treatment (Chambers and Deleo, 2009). Of note, WTAs are exposed at the S. aureus cell surface and they are required for PBP2a-mediated ␤-lactam antibiotic resistance Farha et al, 2013;Maki et al, 1994). Moreover, S. aureus mutants lacking WTA are susceptible to antimicrobial fatty acids from human skin (Kohler et al, 2009) and WTA contributes to S. aureus lysozyme resistance (Bera et al, 2007) suggesting a general protective role of WTA against antimicrobials.…”

Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids

“…Preventing wall teichoic acid (WTA) synthesis by blocking TarO, which catalyzes the first step in the WTA pathway, sensitizes MRSA to β-lactams (11,12). S. aureus WTAs are covalently attached to PG and consist of a poly(ribitol phosphate) [poly (RboP)] backbone containing three tailoring modifications: Dalanylation, α-O-GlcNAcylation, and β-O-GlcNAcylation ( Fig.…”