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The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB.

The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr . These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium , thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d -carboxypeptidases ( d,d -CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d -CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d -CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies. The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.

Vancomycin-resistant enterococci (VRE) have been detected in wild animals representing a public health concern. The red-legged partridge ( Alectoris rufa ) is a common game bird and its meat is consumed in several countries, including Portugal. Three hundred five fecal samples of red-legged partridge from the north of Portugal were screened for VRE. Samples were cultured on Slanetz-Bartley agar supplemented with vancomycin (4 mg/L) and six van A- Enterococcus faecium were recovered. Isolates were tested for antibiotic resistance and virulence genes. Multilocus sequence typing (MLST) was performed to study the genotypic diversity of van A-containing E. faecium . The six isolates showed erythromycin resistance and harbored the erm (B) gene and the four that were tetracycline resistant showed the tet (M) gene. The C-terminal region of the pbp 5 gene of the ampicillin-resistant isolates (minimal inhibitory concentration range of 256 μg/ml) was sequenced. Two different pbp 5 alleles were detected when considering the changes of amino acid in 461–629 region. All isolates harbored the esp gene, whereas hyl , together with the esp gene, was detected in five isolates. MLST analysis grouped the isolates as ST448 ( n  = 1), ST139 ( n  = 1), and ST18 ( n  = 4). Our findings show that the red-legged partridges could be a reservoir of antimicrobial resistance genes and may contribute to the dissemination and transference of the resistance genes to other animals and humans.

Ampicillin and vancomycin are important antibiotics for the therapy of Enterococcus faecalis infections. The ampicillin resistance gene pbp5 is intrinsic in Enterococcus faecium . The vanC1 gene confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum . Both genes are chromosomally located. Resistance to ampicillin and vancomycin was determined in 484 E. faecali s of human and porcine origin by microdilution. Since E. faecalis are highly skilled to acquire resistance genes, all strains were investigated for the presence of pbp5 (and, in positive strains, for the penicillin-binding protein synthesis repressor gene psr ) and vanC1 (and, in positive strains, for vanXYc and vanT ) by using polymerase chain reaction (PCR). One porcine and one human isolate were phenotypically resistant to ampicillin; no strain was vancomycin resistant. Four E. faecalis (3/1 of porcine/human origin) carried pbp5 (MIC=1 mg/L), and four porcine strains were vanC1 positive (minimum inhibitory concentration [MIC]=1 mg/L). Real-time reverse transcriptase (RT)-PCR revealed that the genes were not expressed. The psr gene was absent in the four pbp5 -positive strains; the vanXYc gene was absent in the four vanC1 -positive strains. However, vanT of the vanC gene cluster was detected in two vanC1 -positive strains. To our knowledge, this is the first report on the presence of pbp5 , identical with the “ E. faecium pbp5 gene,” and of vanC1 / vanT in E. faecalis . Even if resistance is not expressed in these strains, this study shows that E. faecalis have a strong ability to acquire resistance genes—and potentially to spread them to other bacteria. Therefore, close monitoring of this species should be continued.

We report on the cloning of the structural gene for penicillin-binding protein 5 (PBP5), lmo2754. We also describe the enzymatic activity of PBP5 and characterize a mutant lacking this activity. Purified PBP5 has dd-carboxypeptidase activity, removing the terminal d-alanine residue from murein pentapeptide side chains. It shows higher activity against low molecular weight monomeric pentapeptide substrates compared to dimeric pentapeptide compound. Similarly, PBP5 preferentially cleaves monomeric pentapeptides present in high-molecular weight murein sacculi. A Listeria monocytogenes mutant lacking functional PBP5 was constructed. Cells of the mutant are viable, showing that the protein is dispensable for growth, but grow slower and have thickened cell walls.