A Family of T6SS Antibacterial Effectors Related to l,d-Transpeptidases Targets the Peptidoglycan.

Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and d-alanine4 and l,d-transpeptidase exchange activity by replacing d-alanine4 by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.

Involvement of penicillin-binding proteins in the metabolism of a bacterial peptidoglycan containing a non-canonical D-amino acid.

Bacteria produce various D-amino acids, including non-canonical D-amino acids, to adapt to environmental changes and overcome a variety of threats. These D-amino acids are largely utilized as components of peptidoglycan, and they promote peptidoglycan remodeling and biofilm disassembly. The biosynthesis, maturation, and recycling of peptidoglycan are catalyzed by penicillin-binding proteins (PBPs). However, although non-canonical D-amino acids are known to be incorporated into peptidoglycan, the maturation and recycling of peptidoglycan containing such residues remain uncharacterized. Therefore, we investigated whether PBP4 and PBP5, low molecular mass (LMM) PBPs from Escherichia coli and Bacillus subtilis, are involved in these events of peptidoglycan metabolism. Enzyme assays using p-nitroaniline (pNA)-derivatized D-amino acids and peptidoglycan-mimicking peptides revealed that PBP4 and PBP5 from both species have peptidase activity toward substrates containing D-Asn, D-His, or D-Trp. These D-amino acids slowed the growth of dacA- or dacB-deficient E. coli (∆dacA or ∆dacB) relative to the wild-type strain. Additionally, these D-amino acids affected biofilm formation by the ∆dacB strain. Collectively, PBP4 and PBP5 are involved in the cleavage of peptidoglycan containing non-canonical D-amino acids, and these properties affect growth and biofilm formation.

Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis.

The peptidoglycan layer of the bacterial cell wall typically contains D-alanine (D-Ala) and D-glutamic acid (D-Glu), and also various non-canonical D-amino acids that have been linked to peptidoglycan remodeling, inhibition of biofilm formation, and triggering of biofilm disassembly. Bacteria produce D-amino acids when adapting to environmental changes as a common survival strategy. In our previous study, we detected non-canonical D-amino acids in Escherichia coli grown in minimal medium. However, the biosynthetic pathways of non-canonical D-amino acids remain poorly understood. In the present study, we identified amino acid racemases in E. coli MG1655 (YgeA) and Bacillus subtilis (RacX) that produce non-canonical D-amino acids other than D-Ala and D-Glu. We characterized their enzymatic properties, and both displayed broad substrate specificity but low catalytic activity. YgeA preferentially catalyzes the racemization of homoserine, while RacX preferentially racemizes arginine, lysine, and ornithine. RacX is dimeric, and appears not to require pyridoxal 5'-phosphate (PLP) as a coenzyme as is the case with YgeA. To our knowledge, this is the first report on PLP-independent amino acid racemases possessing broad substrate specificity in E. coli and B. subtilis.