Molecular and genetic analysis of the Bacteroides uniformis cephalosporinase gene, cblA, encoding the species-specific beta-lactamase.

The gene, cblA, encoding the species-specific, clavulanate-susceptible, endogenous cephalosporinase was cloned from Bacteroides uniformis WAL-7088. The nucleotide sequence was determined, and the cblA structural gene was found to be 891 nucleotides, with a 48% G+C composition, which is similar to that of the B. uniformis genome. The cblA open reading frame encoded an Ambler class A beta-lactamase polypeptide precursor of 296 amino acid residues with a predicted molecular weight of 33,450. A beta-lactamase-deficient B. uniformis mutant with increased beta-lactam susceptibility was constructed by insertional inactivation of the chromosomal gene. This mutant was complemented by plasmids bearing the cblA gene, and the resulting strains were resistant to cephaloridine and had a beta-lactamase that comigrated with the parental beta-lactamase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30,500 Da) and in isoelectric focusing gels (pI 4.6), confirming a role for this beta-lactamase in resistance. Images

In Bacteroides species, ,B-lactamases have been known since the early 1950s (5) and are probably the most important mechanism of resistance to 1-lactam antibiotics (16). The enzymes found among members of this genus show a remarkable degree of diversity and span most of the biochemical classes described by Bush (2,3). These include both cephalosporinases and penicillinases, some of which can hydrolyze cefoxitin or carbapenems. However, the majority ofBacteroides isolates seem to have at least one P-lactamase with activity that is species specific, and these are generally found to be cephalosporinases, which are inhibited by clavulanate, with pIs of from 4.5 to 5.5, and with molecular weights in the range of 30,000 (2,16,21,23,31,33). In order to understand the contributions of these indigenous ,B-lactamases to overall ,-lactam resistance in Bacteroides species, we have begun to characterize the ,-lactamase genes and their regulation. The endogenous ,B-lactamase from Bacteroides fragilis (CepA) was recently cloned and sequenced and was found to belong to Ambler molecular class A (21). Hybridization studies with a cepA gene probe showed that this f-lactamase gene is specific for B. fragilis and that there is little or no nucleotide homology with other Bacteroides species, including Bacteroides uniformis. Thus, it is of interest to determine the relationship among the different Bacteroides ,-lactamases.
In this report we describe the cloning and nucleotide sequence of cblA, which encodes a species-specific P-lactamase in B. uniformis. The gene was most closely related to other Bacteroides ,3-lactamase genes, and the protein belonged to Ambler class A. The cloning was somewhat notable because high-level 3-lactamase expression was obtained in Escherichia coli. Most previous reports of the cloning of Bacteroides antibiotic resistance genes have resulted in low-level or no expression of the resistance phenotype in E. coli (6, 15, 19-21, 25, 26, 32).
Plasmids were transferred in triparental matings from E. coli donors to Bacteroides recipients by standard filter mating protocols (29). These used E. coli donor strains containing RK231 as a conjugation helper plasmid, and the mating plates were incubated aerobically to enhance the growth of donor cells. E. coli transformations were done by the method of Hanahan (7). DNA manipulations and sequence analysis. Routine DNA ligations, endonuclease restrictions, plasmid screening, plasmid purification, agarose gel electrophoresis, and Southern hybridizations were done as described previously (1,13,21). The DNA sequence of the 2.8-kb pFD314 cloned insert was determined in one direction by constructing nested deletions with exonuclease III (8); this was followed by dideoxy sequencing (22) by using Sequenase version 2.0 (United States Biochemical Corp., Cleveland, Ohio). The nucleotide sequence of the opposite DNA strand was elucidated by primer walking by using sequencing primers deduced from the first DNA strand. Analysis of the sequence was performed on a VAX computer by using the GCG analysis software (4), and the GenBank accession number for the cblA sequence is L08472.
The following plasmids were used or constructed for the current study. The standard Bacteroides shuttle and cloning vector pFD288 (Spr CCr) and the suicide vector pFD280.erm (Spr CCr) have been described previously (19,26,29). pFD314 contained the 2.8-kb cblA EcoRI-SmaI fragment cloned into 1711 c Percent activity of nitrocefin assays containing 1 ,uM potassium clavulanate relative to that in assays to which no inhibitor was added. d_ d, assays not performed.
the multiple cloning site of pFD288. The suicide plasmid used for mutagenesis, pFD483, was constructed by insertion of a 560-bp cbL4 HaeIII fragment (base pairs 1071 to 1630) from cblA into the SmaI site of pFD280.erm. In order to perform complementation experiments with the cblA insertion mutant, BU1001flFD483 (Ccr), a tetracycline resistance shuttle vector, was constructed. This plasmid, pFD544, was similar to pFD288 except that the ermF gene conferring Ccr was replaced by a 2.7-kb SstI fragment bearing the Bacteroides tetQ gene (15). The 2.8-kb cblA gene fragment was then inserted to make pFD548, which conferred Spr Apr in E. coli and tetracycline resistance and Apr in B. uniformis.
jI-Lactamase analysis. Cell extracts for I-lactamase assays were prepared in 20 mM sodium phosphate buffer (pH 7.0) with a French pressure cell and were centrifuged at 12,000 x g for 30 min as described previously (19). Activity was measured spectrophotometrically with either nitrocefin (482 nm), benzylpenicillin (233 nm), or cephaloridine (260 nm) as the substrate (17,19), and the results are given in units in which 1 U equals 1 p,mol of substrate degraded min-1 mg of protein-1. Studies testing inhibition with clavulanate were performed by preincubating cell extracts with 1 ,uM clavulanate for 5 min prior to the addition of the nitrocefin substrate as described previously (19). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by using a discontinuous buffer system (11). The 1-lactamase activity in the gels was detected after renaturing the proteins in sodium phosphate buffer by overlaying the gels with nitrocefin-containing agarose (19). The isoelectric focusing gels were run in polyacrylamide gels with ampholytes (pH 3 to 10) exactly as described previously (21).

RESULTS AND DISCUSSION
Cloning and nucleotide sequence ofcbl4. B. uniformis WAL-7088 (Table 1) is a cefoxitin-resistant clinical isolate that uses a combination of mechanisms for ,B-lactam resistance. Wexler and Halebian (35) showed that there are changes in the WAL-7088 penicillin-binding proteins and that the strain produces a cefoxitin-inactivating activity. During our studies of WAL-7088, we demonstrated the presence of the cfxA gene, which encodes a ,-lactamase capable of cefoxitin degradation (19). In addition, a second ,-lactamase is present in this strain, and in order to determine its role in ,-lactam resistance, the gene for this enzyme was cloned and examined. The cloned gene was obtained from a B. uniformis WAL-7088 EcoRI library of about 7,300 clones prepared in E. coli DH5a by using the Spr vector, pFD288. Four Apr colonies were initially observed after plating the library on medium containing 20 ,ug of Ap per ml and 40 ,ug of Sp per ml. Restriction endonuclease analysis of the recombinant plasmids showed that these clones all share a common 7.8-kb EcoRI fragment.
One clone was chosen for further examination, and subcloning experiments indicated that a 2.8-kb EcoRI-SmaI fragment was sufficient to encode Apr in E. coli. Subsequent experiments showed that the Apr phenotype was independent of orientation of the cloned fragment. This DNA fragment present on pFD314 was subjected to exonuclease III treatment, and a set of nested deletions was obtained for DNA sequence determination. Analysis of the nucleotide sequence revealed three open reading frames of greater than 400 bp (Fig. 1). Two of the protein-coding sequences were truncated, but they shared significant homology with predicted proteins of genes in Gen-Bank. The deduced amino acid sequence from the open reading frame designated Env shared 29% identity with the C terminus of the E. coli EnvD protein, which is involved in the cell envelope structure (10). The amino acid sequence deduced from ORF3 was about 31% identical to the AMP nucleosidase predicted from the E. coli amn gene (12). The open reading frame encoding the P-lactamase activity is 891 bp and was designated cblA for chromosomal ,-lactamase (Fig. 2). The cblA structural gene has a G+C content of 48%, which was identical to the composition of the entire cloned 2.8-kb fragment and is consistent with the 46 to 48% G+C content of the B. uniformis species (9). Assignment of the cblA gene was consistent with analysis of the pFD314 deletion derivatives (Fig. 1). The strain with A7, which has its 5' end upstream of the cblA ATG start site, was Apr (MIC, >128 ,ug/ml), but the strain with A2, which has its 5' end within the predicted cblA structural gene, was Aps.
The putative CbIA ,B-lactamase precursor would be 296 amino acid residues, with a predicted molecular weight of 33,450. The relationship of CblA to other ,-lactamases was examined, and the results showed that it is most closely related to two other Bacteroides P-lactamases, forming a unique group of Ambler class A P-lactamases (21). The protein has the greatest homology with the indigenous B. fragilis 3-lactamase, CepA. There was 43% identity between the aligned protein sequences of CepA and CblA, and there was 51% identity between nucleotide sequences of the aligned structural genes for these two peptides. Upstream of the cepA and cblA ATG start sites there was only 36% identity in the nucleic acid sequences for a distance of 500 bp. By comparison, CblA shared just 24% identity with the TEM1 protein and 22% identity with the Bacillus cereus f-lactamase III amino acid sequence.
Analysis and expression of cbUi in B. uniformis. The possibility that cblA encoded the indigenous B. uniformnis chromosomal 1-lactamase was supported by Southern hybridization experiments by using an internal cblA fragment (Fig. 1) as the probe. These results showed that there was a homologous fragment present in EcoRI digests of B. uniformis WAL-7088, V528, and BU1001, but only a very weak hybridization signal was observed with the chromosomes of B. fragilis, Bacteroides ovatus, and Bacteroides vulgatus (27).
Additional evidence for the role of cblA was obtained by constructing a ,B-lactamase-deficient B. uniformis mutant by targeted insertional inactivation. The strain chosen for mutagenesis was BU1001, which contained a cephalosporinase which was inhibited by >50% by 1 ,uM clavulanate (Table 1). For the experiment, an internal cblA gene fragment (Fig. 1) was inserted into the suicide vector pFD280.erm, and the resulting construct, designated pFD483, was then conjugated into BU1001. The clindamycin-resistant transconjugants obtained from this mating were found to have pFD483 inserted into the chromosomal copy of cblA (27). This insertion by a single crossover event was mediated by homologous recombination, and disruption of the gene resulted in a complete loss of 3-lactamase activity, as was seen with strain BUlOOlQl FD483 (Table 1). In addition, there was an 8to 32-fold decrease in the MICs of 1-lactam antibiotics for the mutant BU100lFD483. 13-Lactamase activity could be restored in mutant strains when plasmids containing the cloned cblA gene were introduced into the mutants. This was shown with pFD548, which contained the 2.8-kb EcoRI-Smal cblA fragment (from pFD314) and a tetracycline resistance gene (tetQ) for counterselection. In the mutant BU100WlFD483 containing pFD548, ,B-lactamase activity was restored to nearly the same level as that seen in the parent strain, BU1001. This activity was cephalosporinase in nature and was inhibited by clavulanate, like BU1001 was (Table 1). In contrast, BU1001fl FD483 cells containing only the vector pFD544 displayed no detectable P-lactamase activity, and there was no increase in the P-lactam MICs for those cells (Table 1). Experiments similar to these were attempted with the strain B. uniformis WAL-7088. However, repeated attempts to transfer any plasmids into this strain via conjugation were unsuccessful. The underlying reasons for this inability to function as a recipient strain were not investigated.
Isoelectric focusing and SDS-PAGE were used to characterize the cloned cblA gene product. On the basis of staining of SDS-polyacrylamide gels for nitrocefin activity, strain WAL-7088 had two ,B-lactamases (Fig. 3). One was 38,000 to 40,000 Da and corresponded to CfxA, the ,B-lactamase with cefoxitindegrading activity (19). The second nitrocefin-reactive protein was about 30,500 Da and comigrated with the single band of a protein with P-lactamase activity present in BU1001 cells.  (18), and one study of six strains with a "normal" substrate profile showed that all had a major band with a pl of 4.5 (33). Interestingly, all three bands of proteins with ,B-lactamase activity were missing from the mutant BU1001flFD483 but were restored in mutants containing pFD548 (27). This suggests that there might be some heterogeneity in the processing of the ,-lactamase precursor or some proteolytic degradation of the enzyme upon storage. Results obtained with cell-free extracts of strain WAL-7088 showed that there was consistently one major band of a protein with ,-lactamase activity that comigrated with the band of a protein from BU1001 with 3-lactamase activity (pI 4.6). In addition, a second minor band of a 3-lactamase with a pl of 4.8 was observed. We have shown previously that the CfxA P-lactamase in B. vulgatus CLA341 does not focus well (19). Similarly, results with B. uniformis WAL-7088 demonstrated a large nitrocefin-reactive region in the alkaline region of the gel, but this did not form a definitive band of activity.
Expression of cbUA in E. coli. A variety of studies have demonstrated the differential expression or the lack of expression of Bacteroides antibiotic resistance genes in E. coli (6,20,28,30). The ability of E. coli to express cblA was not the result of a mutation, as was shown previously for the Bacteroides metallo-13-lactamase CcrA (20). This possibility was excluded by transformation experiments with pFD314 in which the antibiotic selection for transformants was either Spr or Apr. The results showed that transformation frequencies were essentially identical regardless of the antibiotic used for selection. If a mutation was required for expression of the B. uniformis cblA gene, the Apr transformation frequency would be much lower than that for spr. Thus, expression of cblA in E. coli must be mediated by a mechanism different from that seen for ccrA.
In summary, the following evidence supports the idea that eblA encodes the species-specific endogenous P-lactamase of B. unifornis. The gene encodes a protein with 3-lactamase activity that comigrates in SDS-polyacrylamide gels and isoelectric focusing gels with the enzyme found in several B. uniformis strains. The enzyme is a cephalosporinase, it is susceptible to clavulanate, and it has a pI of 4.6, all of which are characteristics similar to those previously reported for the low-level, endogenous B. unifornis f-lactamases (Table 1) (18,31,33). Hybridization studies showed that cblA is homologous to DNA fragments in B. uniformis but not other Bacteroides species. Finally, B. uniformis mutants lacking 1-lactamase were constructed by using a cbl4 gene fragment to mediate insertional inactivation of the gene.