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Xanthomonas campestris pv. campestris is a gram-negative phytopathogenic bacterium causing black rot in crucifers (25). In a previous study it was found that among 86 strains of X. campestris pv. campestris isolated in Taiwan, 81 were resistant to ampicillin at a level of 50 µg/ml (12a). Antibiotic resistance can be transferred by a bacterium through a variety of mechanisms, including transduction, conjugation, or transfection, and once a resistance gene is transferred, it may be incorporated into the genome or plasmids of the recipient bacterium (16). Since the genus Xanthomonas inhabits soil, infected plants, plant debris, and asymptomatic plants near acutely infected plants, the possibility of widespread ampicillin resistance in this genus raises environmental concerns. In this study, a periplasmic -lactamase was detected in X. campestris pv. campestris strain 11. The amino acid sequence deduced from the bla gene revealed a high degree of similarity between the -lactamase of strain 11 and the L2 serine -lactamase of Stenotrophomonas maltophilia (22).

Bacterial strains, growth conditions, and DNA techniques. X. campestris pv. campestris strains 11, 11A, and 17 have been described (26). The other Xanthomonas strains were provided by S.-T. Hsu. Luria-Bertani (LB) medium and L agar (13) were used as general-purpose media with or without ampicillin (50 µg/ml). X. campestris was cultivated at 28°C. The DNA techniques employed were those described by Sambrook et al. (18). DNA sequences were determined by the method of Sanger et al. (19).

Detection of -lactamase in strain 11. To determine the cellular location of the -lactamase, strain 11 cells were fractionated by the method of Osborn and Munson (15), with minor modifications. Proteins from different fractions were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Detection of -lactamase activity in situ was carried out with benzylpenicillin (100 µg/ml) as the substrate, as described by Tai et al. (21). A single protein band in the periplasmic fraction was found to contain -lactamase activity and was not detected in cytoplasmic, membrane, and extracellular fractions (data not shown). This enzyme, with a molecular mass of 30 kDa as determined by gel electrophoresis, has a size similar to those of other class A -lactamases detected in gram-negative organisms (3, 8, 20, 22), suggesting that the 30-kDa protein is indeed a -lactamase.

Antibiotic sensitivity of strain 11. We carried out MIC determinations according to the microdilution method recommended by the National Committee for Clinical Laboratory Standards, using -lactams purchased from Sigma (St. Louis, Mo.). The MICs determined were as follows (in micrograms per milliliter): cephaloridine, 128; cephalothin, 256; penicillin, 64; ampicillin, 128; carbenicillin; 128; cefotaxime, 8; cefoxitin, 16; oxacillin, 256; and piperacillin, 128. Results indicated that strain 11 was highly resistant to the penicillins and narrow-spectrum cephalosporins used in this study, was less resistant to cefoxitin (an expanded-spectrum cephalosporin), and was susceptible to cefotaxime (a broad-spectrum cephalosporin).

Cloning and sequencing of the strain 11 bla gene. The strain 11 bla gene was cloned, by conferring ampicillin resistance in E. coli DH5, from a genomic library constructed with pBK-CMV. Deletion mapping located the bla gene in the 1.7-kb SstI-EcoRI fragment (Fig. 1).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Localization of the -lactamase gene of X. campestris pv. campestris strain 11. Plasmid pBK-1 contains a 4.5-kb Sau3A1 insert cloned into the multiple cloning sites of vector pBK-CMV. For deletion mapping, the 3.1-kb SstI-HindIII fragment, the 1.7-kb SstI-EcoRI fragment, the 0.8-kb SstI-XhoI fragment, and the 0.9-kb XhoI-EcoRI fragment from the pBK-1 insert were subcloned into the compatible sites of pBK-CMV to generate pBKH, pBKE, pBKX, and pBKXE, respectively. +, transformants resistant to ampicillin; , transformants sensitive to ampicillin.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence (1,190 bp) of the upstream region of the pBKE insert. The deduced amino acids are represented by one-letter codes. Initiation and stop codons are boldfaced. The underlined regions (bp 1114 to 1139 and bp 1142 to 1167) are inverted repeats which potentially form a stem-loop structure resembling a transcription termination signal. Boldfaced letters in the repeats (16 out of 26 nucleotides in each region) represent the complementary bases. SD, ribosome-binding sequence.

Sequence comparison and classification of the strain 11 -lactamase. Several schemes have been proposed for the classification of bacterial -lactamases. Ambler (1) suggested a scheme containing four classes of -lactamases based on primary amino acid sequences. The most recent functional scheme by Bush et al. (5) considered the inhibition characteristics, substrate profiles, and molecular structures of the enzymes. Based on these parameters, they constructed a dendrogram showing relationships among 88 -lactamases. In this study, we constructed a dendrogram that clusters the strain 11 -lactamase with 20 other related sequences, including sequences which were not included in the dendrogram of Bush et al. (5). The highest degree of relatedness to the strain 11 -lactamase was found in the L2 serine -lactamase from S. maltophilia, an Ambler class A enzyme which falls in group 2e of the Bush scheme (Fig. 3). Consistent with this finding, the strain 11 -lactamase has the highest degree of identity (51.5%) with S. maltophilia L2 (22) and lower degrees with several other class A/group 2 enzymes: Yersinia enterocolitica BLA-A (45.0%), E. coli TOHO-1 (44.5%), and E. coli MEN-1 (44.5%) (2, 3, 8, 20) (Fig. 3).

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Dendrogram of -lactamases based on the sequence similarity between the strain 11 -lactamase and the 20 other sequences most closely related. The signal sequences were not included for comparison. Asterisks indicate the sequences not included in the dendrogram of Bush et al. (5). The Genetics Computer Group package was used for the construction.

View larger version (78K): [in this window] [in a new window]   FIG. 4.   Alignment of the deduced amino acid sequences of the class A -lactamases from gram-negative bacteria S. maltophilia L2 (22), Y. enterocolitica BLA-A (20), E. coli TOHO-1 (8), and E. coli MEN-1 (2, 3). The consensus line displays the residues conserved in the sequences, with the boldfaced letters indicating the residues conserved in the strain 11 -lactamase. The consensus sequences STXK, SDN, KTG and the glutamic acid residue located 34 residues behind the SDN loop are underlined. The Genetics Computer Group package was used in these analyses.

Northern blot analysis. The 317-bp XhoI-NotI fragment (bp 734 to 1050; Fig. 2) within the strain 11 bla gene was used as the probe for Northern hybridization with the RNA prepared from strain 11 cells grown in LB medium and treated with ampicillin 30 min prior to RNA extraction (24). A hybridization band of ca. 980 nt, with smearing in the lower area of the gel, was detected (data not shown). This transcript, having a size similar to that of the bla gene, appears to be monocistronic.

Construction of the bla mutant strain 11bla::Tc and complementation test. Mutation in strain 11 bla was performed according to Lee et al. (10). A 2.0-kb tetracycline resistance (Tc^r) cartridge from mini-Tn5Tc (7) was inserted into the SmaI site of the pBKE insert. The interrupted bla gene was electroporated into strain 11 (23), followed by replacement of the chromosomal wild-type version. One of the tetracycline-resistant (15 µg/ml) and ampicillin-sensitive mutants (strain 11bla::Tc) thus obtained was verified by Southern hybridization to have undergone double crossover.

Distribution of bla gene among xanthomonads. Thirteen other strains of X. campestris, representing eight pathovars and one Xanthomonas oryzae pv. oryzae strain, were tested for resistance by being grown on LB agar containing ampicillin. All strains except X. campestris pv. mangiferaeindicae strain 38 were found to be ampicillin resistant. Southern hybridization revealed that for each of the EcoRI fragments of chromosomal DNA from these X. campestris strains, except strain 38, for which no signal was observed, a single band was detected. A 3.8-kb fragment was detected in X. campestris pathovars campestris (strains 2, 6, 11A, 17, and 85), begoniae (strain 59), dieffenbachiae (strain 65), and phaseoli (strain 73); a 10-kb fragment was detected in pathovars citri (strain 60), glycines (strain 69), and vesicatoria (strain 64); and a 5.5-kb fragment was detected in X. oryzae pv. oryzae (strain 21). Since only one fragment was detected, it is likely that only one copy of the bla gene is present in each of these Ap^r strains. These results indicate that the bla genes are highly homologous and widespread among xanthomonads.

Conclusion. In this study, we have identified a novel -lactamase from X. campestris, a plant-pathogenic bacterial species presently not involved in human or animal pathology. In addition, the presence of a -lactamase gene has been demonstrated to be widespread among xanthomonads. It is thought that unusual -lactamases produced by human pathogens are acquired from some other sources; the occurrence of this enzyme in the botanical world suggests one of the possible novel origins.

Nucleotide sequence accession number. The nucleotide sequence determined here has been deposited in GenBank under accession no. AF091319.