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. 2004 Feb;48(2):484-90.
doi: 10.1128/aac.48.2.484-490.2004.

The ybxI Gene of Bacillus Subtilis 168 Encodes a Class D Beta-Lactamase of Low Activity

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Free PMC article

The ybxI Gene of Bacillus Subtilis 168 Encodes a Class D Beta-Lactamase of Low Activity

Maria-Luigi Colombo et al. Antimicrob Agents Chemother. .
Free PMC article

Abstract

The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase.

Figures

FIG. 1.
FIG. 1.
Interaction between the serine PREs with β-lactam antibiotics. k2/K and k3 are the acylation and the deacylation rate constants, respectively. For β-lactamases, k3 is large (≥1,000 s−1 for good substrates), and for dd-peptidases and PBPs k3 is low (≤10−3 s−1).
FIG.2.
FIG.2.
Sequence alignment of class D β-lactamases and C-terminal domains of penicillin receptors. Alignment of amino acid sequences of the YbxI protein; class D β-lactamases OXA-20 (30), OXA-2 (7), OXA-15 (8), OXA-3 (37), PSE2 (19), OXA-11 (16), OXA-7 (38), OXA-5 (6), LCR1 (6), OXA-1 (32), OXA-18 (34), and OXA-9 (40); and the CTDs of penicillin receptors involved in the induction of β-lactamases (B. licheniformis [B.li] BlaR [26] and S. aureus [S.au] BlaR [35] or methicillin-resistant S. aureus PBP [S. aureus MecR] [18]). The underlined sequence in YbxI corresponds to the signal peptide. In the consensus sequence, residues in uppercase letters are strictly conserved in all aligned sequences. The residues in lowercase letters are those conserved in more than 50% of the sequences. The other consensus symbols are as follows: ! is either I or V; $ is L or M; % is F or Y; and # is N, D, Q, or E. The residues involved in the active-site signatures of serine-PREs are underlined. Dots indicate deletions. Amino acid sequence alignments were performed with the Multalin program of the ExPAsy server (http://us.expasy.org/tools/#align) (5).
FIG. 3.
FIG. 3.
SDS-PAGE analysis of crude cellular extracts and at different stages of YbxI purification. Lanes 1 and 2, crude cellular extracts of noninduced and induced (0.5 mM IPTG) E. coli/pCIP 51, respectively; lane 3, periplasmic fraction after cell lysis; lanes 4 to 6, YbxI after Source 15Q, Source 15S, and Sephacryl S100 chromatography steps, respectively; MM, molecular mass markers. The arrow indicates the YbxI position.
FIG. 4.
FIG. 4.
Effect of bicarbonate on the rate of nitrocefin hydrolysis. The experiments were carried out in phosphate buffer (pH 7.0) that was degassed (○) or not degassed (□), with addition of 0.150 (▵), 3 (•), 6 (⋄), 25 (×), and 50 (♦) mM bicarbonate and with addition of 50 mM NaCl (▪). Initial rate (v0) values were determined with 0.07 μM YbxI in a total volume of 500 μl. Error bars, standard deviations.
FIG. 5.
FIG. 5.
Time course analysis of YbxI acylation by fluorescent ampicillin (Ampi-flu). SDS-PAGE and fluorography of YbxI (85 pmol) after incubation (0, 10, 20, 40, 60, and 80 min) with Ampi-flu (500 pmol). BlaR-CTD (25 kDa) (400 pmol) was used as a positive control and was incubated with Ampi-flu for 15 min.
FIG. 6.
FIG. 6.
RT-PCR analysis of ybxI transcripts in B. subtilis. RT-PCR analysis by agarose gel electrophoresis of ybxI transcripts in B. subtilis in the presence or in absence of a β-lactam stress compound. Abbreviations: NS, nonstressed cells; S, cells stressed by the presence of 2.5 μg of cephalosporin C; MM,: molecular mass marker. Lanes 1 to 4, yjbJ control; lane 1, RT-PCR NS; lane 2, PCR NS; lane 3, RT-PCR S; lane 4, PCR S; Lanes 5 to 8, ybxI; lane 5, RT-PCR NS; lane 6, PCR NS; lane 7, RT PCR S; lane 8, PCR S; Lanes 9 and 10 correspond to the PCR products of ybxI and yjbJ, respectively, obtained with the genomic DNA of B. subtilis 168.

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