Cloning and sequencing of the low-affinity penicillin-binding protein 3r-encoding gene of Enterococcus hirae S185: modular design and structural organization of the protein

Piras, Graziella; Raze, Dominique; Kharroubi, Aboubaker El; Hastir, D; Englebert, S.; Coyette, Jacques; Ghuysen, Jean‐Marie

doi:10.1128/jb.175.10.2844-2852.1993

Cited by 41 publications

(42 citation statements)

References 24 publications

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“…The genes of different strains of three species coding for these PBPs have been cloned and sequenced (13,14,31,33,38,45). Moreover, with the DNA sequencing data collected recently from clones of E. hirae, E. faecalis, and E. faecium (13, 45; this study) and the determination of the genomic sequences of E. faecalis V583 (http://tigr.org) and E. faecium DO (http://genome.ornl.gov/microbial/efae/), it is possible to establish the genetic context of these genes.…”

Section: Discussionmentioning

confidence: 99%

Redefining the Role of psr in β-Lactam Resistance and Cell Autolysis of Enterococcus hirae

et al. 2003

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The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the ␤-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to ␤-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in ␤-lactam resistance or in the regulation of cell autolysis in E. hirae.The natural low susceptibility of enterococci to ␤-lactams is due to a low-affinity penicillin-binding protein (PBP) that is overproduced in moderately resistant laboratory mutants and some clinical strains (16,17,35,45). Highly resistant clinical strains generally do not overproduce this low-affinity PBP but modify its primary structure to further reduce its binding capacity (21,24,35,45). Conversely, when the low-affinity PBP is not synthesized, the cells become highly susceptible to benzylpenicillin (PenG) (18).Sequencing of the enterococcal genes encoding low-affinity PBPs showed that these proteins are relatively similar (ϳ75 kDa) (13,14,33,35,45). Including PBP2Ј of methicillin-resistant staphylococci (1) and PBP3 of Bacillus subtilis (28), these protein form subgroup B1 of the class B high-molecular-mass PBPs (19). When penicillin is present, they can take over the functions of most if not all the other PBPs. This has not been verified for PBP3 in B. subtilis. Thus, in enterococci and staphylococci these proteins appear to be multifunctional PBPs that are not essential for growth under laboratory conditions but synthesize peptidoglycan when the other PBPs are inhibited (5,6,7,17,18).The study of Enterococcus hirae contributed a great deal to these observations. From wild-type strain ATCC 9790 (MIC of PenG, 0.6 g/ml), resistant strain R40 (MIC of PenG, 60 g/ml) was isolated by a four-step selection procedure on plates containing increasing PenG concentrations. The greater resistance of strain R40 was attributed to overproduction of the low-affinity PBP5 (17). However, it also appeared that R40 differed from parent strain ATCC 9790 by slower growth, faster autolysis, a lower cell wall rhamnose content, and greater susceptibility to lysozyme (27). PenG-hypersusceptible strain Rev14 (MIC of PenG, 0.015 g/ml), which does not synthesize PBP5, was derived from R40 by chemical mutagenesis (18). Except for its high level of susceptibility to ␤-lactams, this strain has the properties described above for R40 (27).Expression of the PBP5-encoding gene, pbp5, in E. hirae was proposed to be under control of psr...

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Section: Discussionmentioning

confidence: 99%

Redefining the Role of psr in β-Lactam Resistance and Cell Autolysis of Enterococcus hirae

et al. 2003

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“…Inactivation of PBP5 of Enterococcus faecalis leads to hypersusceptibility to all ␤-lactams (93). Interestingly, the first low-affinity PBP found to be located on a plasmid is PBP3r from Enterococcus hirae, which is almost identical to PBP5 (80,82).…”

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confidence: 98%

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

Rohrer

Berger‐Bächi

2003

Antimicrob Agents Chemother

108

102

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“…The mecA gene and the associated large (40-to 60-kb) mec element (9,10,13,15,21,27) are not native to S. aureus but were acquired from an extraspecies source by an unknown mechanism (3, 18). The nature of the extraspecies source, i.e., the evolutionary origin of mecA and the formation of the mec element, has remained largely a matter of speculation (1,8,11,20,26).In a recent effort to track the evolutionary origin of mecA, we used a DNA probe internal to this gene in S. aureus to screen bacterial isolates belonging to 13 different staphylococcal species for bacteria that would give a positive signal with this DNA probe under hybridization conditions of high stringency. This effort has led to the identification of a close homologue of the S. aureus mecA gene in Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found mainly in rodents and primitive mammals (4).…”

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Recruitment of the mecA Gene Homologue of Staphylococcus sciuri into a Resistance Determinant and Expression of the Resistant Phenotype in Staphylococcus aureus

2001

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Strains of methicillin-resistant Staphylococcus aureus (MRSA) have become the most important causative agents of hospital-acquired diseases worldwide. The genetic determinant of resistance, mecA, is not a gene native to S. aureus but was acquired from an extraspecies source by an unknown mechanism. We recently identified a close homologue of this gene in isolates of Staphylococcus sciuri, a taxonomically primitive staphylococcal species recovered most frequently from rodents and primitive mammals. In spite of the close sequence similarity between the mecA homologue of S. sciuri and the antibiotic resistance determinant mecA of S. aureus, S. sciuri strains were found to be uniformly susceptible to ␤-lactam antibiotics. In an attempt to activate the apparently "silent" mecA gene of S. sciuri, a methicillin-resistant derivative, K1M200 (for which the MIC of methicillin is 200 g/ml), was obtained through stepwise exposure of the parental strain S. sciuri K1 (methicillin MIC of 4 g/ml) to increasing concentrations of methicillin. DNA sequencing of the mecA homologue from K1M200 revealed the introduction of a point mutation into the ؊10 consensus of the promoter: the replacement of a thymine residue at nucleotide 1577 in the susceptible strain K1 by adenine in the resistant strain K1M200, which was accompanied by a drastic increase in transcription rate and the appearance of a new protein that reacted with monoclonal antibody prepared against the penicillin-binding protein 2A (PBP2A), i.e., the gene product of S. aureus mecA. Transduction of mecA from K1M200 (cloned into a plasmid vector) into a methicillin-susceptible S. aureus mutant resulted in a significant increase of methicillin resistance (from a methicillin MIC of 4 g/ml to 12 and up to 50 g/ml), the appearance of a low-affinity PBP detectable by the fluorographic assay, and the production of a protein that reacted in a Western blot with monoclonal antibody to PBP2A. Antibiotic resistance and the protein products disappeared upon removal of the plasmid-borne mecA homologue. The observations support the proposition that the mecA homologue ubiquitous in the antibiotic-susceptible animal species S. sciuri may be an evolutionary precursor of the methicillin resistance gene mecA of the pathogenic strains of MRSA.The emergence and worldwide spread of methicillin-resistant Staphylococcus aureus (MRSA) between the early 1960s and the late 1990s have begun to pose serious threats to the chemotherapy of staphylococcal diseases worldwide. The genetic determinant of methicillin resistance in MRSA is the acquired gene mecA, which encodes the low-affinity penicillinbinding protein 2A (PBP2A), which, according to current theory, can function as a surrogate transpeptidase in the presence of high concentrations of ␤-lactam antibiotics that inactivate the four high-affinity PBPs native to S. aureus (5). The mecA gene and the associated large (40-to 60-kb) mec element (9,10,13,15,21,27) are not native to S. aureus but were acquired from an extraspecies source by an unknown mecha...

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Cloning and sequencing of the low-affinity penicillin-binding protein 3r-encoding gene of Enterococcus hirae S185: modular design and structural organization of the protein

Cited by 41 publications

References 24 publications

Redefining the Role of psr in β-Lactam Resistance and Cell Autolysis of Enterococcus hirae

Redefining the Role of psr in β-Lactam Resistance and Cell Autolysis of Enterococcus hirae

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

Recruitment of the mecA Gene Homologue of Staphylococcus sciuri into a Resistance Determinant and Expression of the Resistant Phenotype in Staphylococcus aureus

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