TheybxIGene ofBacillus subtilis168 Encodes a Class D β-Lactamase of Low Activity

Abstract

Two families of bacterial serine enzymes recognize penicillin and other ␤-lactam antibiotics and form a superfamily of evolutionarily related proteins named serine penicillin-recognizing enzymes (PREs) (21). The first family contains the D-alanyl-D-alanine peptidases (DD-transpeptidases) involved in the biosynthesis or in the control of the cross-linking of the peptidoglycan, the main constituent of the bacterial cell wall. DD-peptidases are inactivated by ␤-lactam antibiotics, as a result of the acylation of their active serine by the antibiotic, which yields a very stable covalent adduct (Fig. 1). Because of these properties, these enzymes are also referred to as penicillin-binding proteins (PBPs) (10). The members of the second family, the serine ␤-lactamases are subdivided into three classes (A, C, and D) and efficiently hydrolyze the amide bond of the ␤-lactam nucleus, to yield products devoid of antibiotic activity (12, 31). For serine ␤-lactamases, the catalytic pathway involves the formation of an acyl-enzyme similar to that obtained in the interaction of the ␤-lactam antibiotic with the DD-peptidases, but in the case of ␤-lactamases the acyl-enzyme is very unstable. Although one might think that they would be very different, the two families of enzymes share many biochemical characteristics and very similar three-dimensional (3D) structures (17,22,33,36). Along their primary structures, three conserved peptide sequences, important for recognition of the substrate or catalysis, have been identified by comparison of their 3D structures (Table 1). Sequence identities between the class D and the class A and C enzymes are on average 16% (29). Nevertheless, these proteins are thought to have a common evolutionary history (15,21,24).By database comparison, three open reading frames, penP, ybbE, and ybxI, have been found in the B. subtilis 168 genome which encode putative ␤-lactamases. PenP has been overproduced and characterized as a class A ␤-lactamase (J. M. van Dijl, unpublished data), whereas YbbE remains uncharacterized, and YbxI has been postulated to be a class D ␤-lactamase because its primary structure exhibits a high similarity with the members of this group of serine ␤-lactamases. A phylogenetic tree was constructed by Barlow and Hall (3), in which the ybxI nucleotide sequence was referred to as B. subtilis z99105. No promoter sequence was identified upstream of the coding sequences for these three genes, in agreement with the fact that no ␤-lactamase activity was detected in growing B. subtilis 168 cells or in the supernatant. To determine if YbxI is a class D ␤-lactamase, it was overproduced in the periplasm of Escherichia coli, purified to homogeneity, and analyzed for its capacity to hydrolyze ␤-lactam antibiotics and DD-peptidase substrates. YbxI was shown to be a ␤-lactamase of very low activity which is not inactivated by clavulanic acid. Indeed, the recombinant protein interacts with several ␤-lactam antibiotics and can hydrolyze some of them but does not hydrolyze the peptides cleaved by DD...

Analysis of the role ofBacillus subtilisσMin β‐lactam resistance reveals an essential role for c‐di‐AMP in peptidoglycan homeostasis

“…B. subtilis displays a significant level of intrinsic resistance against a variety of β‐lactam antibiotics, but the underlying mechanisms are poorly understood. Although there are three putative β‐lactamase genes ( penP , ybbE and yblX ) in the genome, no β‐lactamase activity is detected in the growing cells or supernatant (Colombo et al ., 2004). No penicillin‐insensitive PBP alleles have been identified nor does an efflux pump‐based mechanism appear to be applicable to B. subtilis and other Gram‐positive bacteria.…”

Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii

“…In addition to the class A BCL-type ␤-lactamase PenP, in silico analysis of the genome of B. clausii KSM-K16 (GenBank accession no. YP_173769) revealed the presence of two genes encoding class D ␤-lactamases sharing 54% and 35% amino acid identity with YbxI from B. subtilis (12). Nevertheless, the amino acid sequence of the first of these two sequences lacks the catalytic lysine residue of the classical SXXK motif.…”

“…Further studies will be necessary to show if these class D ␤-lactamases significantly contribute to the overall ␤-lactam susceptibility pattern of B. clausii. Indeed, Colombo et al reported that YbxI of B. subtilis had a hydrolytic activity intermediate between those of penicillin-binding proteins and ␤-lactamases (12). Genomic analysis of B. clausii KSM-K16 showed that it does not seem to contain additional chromosomally encoded metallo-␤-lactamase in contrast to B. anthracis, which contains a gene encoding Bla2 (class B) in addition to a gene for Bla1 (class A) (9), or B. cereus, which contains a gene for BcII (class B) in addition to the BcI and BcIII (class A) genes (9,13).…”

An active β‐lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species

“…The B. subtilis genome encodes two β-lactamases -YbxI and PenP. For the putative class D β-lactamase YbxI, a reverse transcription RT-PCR analysis did not detect the presence of mRNA encoding YbxI during exponential phase of growth of the strain B. subtilis 168, suggesting that YbxI was not expressed under these conditions and in that strain (Colombo et al, 2004). However, when recombinant YbxI was expressed and purified in E. coli, the protein was acylated by several β-lactam antibiotics and hydrolysed some of them, but at a very low rate (Toth et al, 2016).…”

“…However, when recombinant YbxI was expressed and purified in E. coli, the protein was acylated by several β-lactam antibiotics and hydrolysed some of them, but at a very low rate (Toth et al, 2016). The conditions under which the enzyme YbxI is activated and its exact role in the undomesticated B. subtlis 3610 remain unknown (Colombo et al, 2004;Toth et al, 2016). In contrast, the class A β-lactamase PenP has been shown to be secreted (Hirose et al, 2000) and enzymatically active when overexpressed (Colombo et al, 2004).…”