A metallo-β-lactamase enzyme for internal detoxification of the antibiotic thienamycin

AbstractThienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

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Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

10.1101/2021.02.12.430937 ◽

2021 ◽

Author(s):

Henri Voedts ◽

Delphine Dorchêne ◽

Adam Lodge ◽

Waldemar Vollmer ◽

Michel Arthur ◽

...

Keyword(s):

Turgor Pressure ◽

Cross Linking ◽

Penicillin Binding Proteins ◽

Peptidoglycan Synthesis ◽

Dual Specificity ◽

Common Motif ◽

Cross Links ◽

Hydrolysis Of ◽

Controlled Hydrolysis

ABSTRACTBacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

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Roles of ATP Hydrolysis by FtsEX and Interaction with FtsA in Regulation of Septal Peptidoglycan Synthesis and Hydrolysis

mBio ◽

10.1128/mbio.01247-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Shishen Du ◽

Sebastien Pichoff ◽

Joe Lutkenhaus

Keyword(s):

Cell Separation ◽

Atp Hydrolysis ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Separation System ◽

Content Type ◽

E Coli ◽

Peptidoglycan Synthesis ◽

Hydrolysis Of

ABSTRACT In Escherichia coli, FtsEX coordinates peptidoglycan (PG) synthesis and hydrolysis at the septum. It acts on FtsA in the cytoplasm to promote recruitment of septal PG synthetases and recruits EnvC, an activator of septal PG hydrolases, in the periplasm. Following recruitment, ATP hydrolysis by FtsEX is thought to regulate both PG synthesis and hydrolysis, but how it does this is not well understood. Here, we show that an ATPase mutant of FtsEX blocks septal PG synthesis similarly to cephalexin, suggesting that ATP hydrolysis by FtsEX is required throughout septation. Using mutants that uncouple the roles of FtsEX in septal PG synthesis and hydrolysis, we find that recruitment of EnvC to the septum by FtsEX, but not ATP hydrolysis, is required to promote cell separation when the NlpD-mediated cell separation system is present. However, ATP hydrolysis by FtsEX becomes necessary for efficient cell separation when the NlpD system is inactivated, suggesting that the ATPase activity of FtsEX is required for optimal activity of EnvC. Importantly, under conditions that suppress the role of FtsEX in cell division, disruption of the FtsEX-FtsA interaction delays cell separation, highlighting the importance of this interaction in coupling the cell separation system with the septal PG synthetic complex. IMPORTANCE Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. In E. coli, the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

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