ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2004, p. 209–215 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.1.209–215.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 1
Constitutive Expression of a Chromosomal Class A (BJM Group 2) -Lactamase in Xanthomonas campestris Shu-Fen Weng,1 Juey-Wen Lin,2 Chih-Hung Chen,1 Yih-Yuan Chen,1 Yi-Hsuan Tseng,1 and Yi-Hsiung Tseng1* Institute of Molecular Biology1 and Institute of Biochemistry,2 National Chung Hsing University, Taichung 402, Taiwan, Republic of China Received 28 February 2003/Returned for modification 29 April 2003/Accepted 23 September 2003
Sequencing of the upstream region of the -lactamase gene from Xanthomonas campestris pv. campestris 11 (blaXCC-1) revealed the cognate ampR1 gene (289 amino acids, 31 kDa). It runs divergently from blaXCC-1 with a 100-bp intergenic region (IG) containing partially overlapped promoters with structural features typical of the bla-ampR IG. The deduced AmpR1 protein shows significant identity in amino acid sequence and conserved motifs with AmpR proteins of other species, e.g., of Pseudomonas aeruginosa (58.2% amino acid identity). Results of insertional mutation, complementation tests, and -lactamase assays suggested that expression of blaXCC-1 was constitutive and dependent on AmpR1. Four bla genes and two ampR genes are present in the fully sequenced X. campestris pv. campestris ATCC 33913 genome, with XCC3039 and XCC3040 considered the analogues of blaXCC-1 and ampR1, respectively. An ampR1 homologue was detected by Southern hybridization in the ampicillin-resistant Xanthomonas strains, which appear to express -lactamase constitutively. Although the significance remains to be studied, constitutive expression of -lactamase by a widespread bacterial genus raises environmental concerns regarding the dissemination of resistance genes. ampC. AmpG (a permease) and AmpD (a cytosolic amidase) are enzymes involved in cell wall metabolism, required for the transport of murein breakdown products (muropeptides) into the cytoplasm (12). AmpD negatively regulates ampC expression, while AmpG is required for activation of ampC by AmpR. In the chromosomal ampR-ampC systems characterized, the ampC genes are usually inducible (1, 2, 17, 21). The enhanced expression of ampC in Enterobacter, Citrobacter, and Pseudomonas strains with both hyperinducible and high-level constitutive -lactamase production has been shown to be due to a mutation in ampD (5, 13). In this study, we cloned and sequenced the DNA region upstream of the blaXCC-1 gene, revealing the entire ampR1 gene, and tested the effects of mutation in ampR1 on the expression of the blaXCC-1 gene. The results showed that the blaXCC-1 gene appears to be expressed constitutively, requiring the function of AmpR1. All other ampicillin-resistant Xanthomonas strains tested also appear to express -lactamase constitutively.
Xanthomonas campestris pv. campestris is a gram-negative phytopathogenic bacterium causing black rot in crucifers (32). In our previous study, it was found that strains of Xanthomonas isolated in Taiwan are commonly resistant to ampicillin at a level of 50 g/ml (29). To characterize the resistance property, a bla gene (renamed blaXCC-1 in this study) coding for a periplasmic -lactamase of 30 kDa in X. campestris pv. campestris 11 (strain Xc11) was cloned and sequenced (6, 29). Its deduced amino acid sequence shows identity (51.5%) and conserved domains to the Stenotrophomonas maltophilia L2 and the other Ambler class A or Bush group 2 -lactamases. Southern hybridization using the blaXCC-1-specific probe showed that homologous -lactamase genes were widespread in xanthomonads. In addition, sequencing data have also revealed the N-terminal 41 codons of the regulatory ampR gene (designated ampR1) in the upstream region of blaXCC-1 (29). The expression of -lactamase genes (ampC) in members of the family Enterobacteriaceae is under the control of three genes, ampR, ampD, and ampG (3, 10, 12, 19, 23). The ampR gene is usually located immediately upstream of the cognate -lactamase gene (ampC) and transcribed divergently, encoding a trans-acting protein (AmpR) which belongs to the LysR family of bacterial regulators (26). In Citrobacter freundii, AmpR has been shown to bind to a 38-bp sequence within the intergenic region (IG) between ampR and ampC (16). It was suggested that in the inactive state, AmpR acts as a weak repressor of ampR transcription, causing a negative autoregulation as in many similar regulatory loci, whereas in the activated state, it acts as a strong activator for the transcription of
MATERIALS AND METHODS Bacterial strains and growth conditions. The strains used in this study are listed in Table 1. Luria-Bertani (LB) medium and L agar (18) were used as the general-purpose media for cultivation of Xanthomonas (28°C), Serratia (37°C), and Escherichia coli (37°C). Antibiotics were added when necessary: ampicillin (50 g/ml), kanamycin (50 g/ml), and tetracycline (15 g/ml). Construction of plasmids. Plasmids carrying inserts from the X. campestris pv. campestris 11 (strain Xc11) chromosome are depicted in Fig. 1. Plasmids pBKE and pRKSE were previously constructed by cloning the 1.7-kb EcoRI-SstI fragment including the blaXCC-1 gene and the N-terminal region of ampR1 into the E. coli vector pOK12 and the broad-host-range vector pRK415, respectively (29). pBKX was obtained by cloning the downstream PstI-SstI fragment from the pBKE insert into pOK12 (27). Using the labeled pBKX insert for hybridization, pCH1 (with a ca. 4.0-kb insert) was obtained from a library constructed by cloning the Sau3A1 fragments from the Xc11 chromosome into pOK12. Plasmid pFY13-9BSRX carried a 2.5-kb insert from pCH1 containing the entire ampR1 and blaXCC-1 genes, cloned in the XbaI-StuI sites of pFY13-9 (14). Plasmid
* Corresponding author. Mailing address: Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan, Republic of China. Phone: 886-4-2285-1885. Fax: 886-4-2287-4879. E-mail:
[email protected]. 209
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Strain
E. coli DH5␣ ATCC 25922 E. coli(pFY13-9BSRX) E. coli(pBX) E. coli(pRX) X. campestris pv. campestris Xc2 Xc6 Xc85 Xc17 Xc11 Xc11A Xc11bla::Tc Xc11RM Xc11bla::Tc(pRKSE) Xc11RM(pFJ-ampR1) X. X. X. X. X. X. X. X.
campestris pv. begoniae 59 campestris pv. dieffenbachiae 65 campestris pv. mangiferaeindcae 38 campestris pv. phaseoli 73 axonopodis pv. citri 60 axonopodis pv. glycines 69 oryzae pv. oryzae 21 axonopodis pv. vesicatoria Xv64 Xv3240 Serratia marcescens 6 a
Characteristic(s)
endA1 hsdR17 (rk⫺ mk⫹) supE44 thi-1 recA gyrA relA1 80dlacZ⌬M15 ⌬(lacZYA-argF) Reference strain for MIC tests DH5␣ with plasmid pFY13-9BSRX carrying the entire ampR1 and blaXCC-1 genes, Kmr/Aps DH5␣ with plasmid pBX carrying the blaXCC-1 promoter situated upstream of the luxA and fluxB reporter genes, Tcr/Aps DH5␣ with plasmid pRX carrying the ampR1 promoter situated upstream of the luxA and luxB reporter genes, Tcr/Aps
Source or reference
Bethesda Research Laboratories American Type Culture Collection This study This study This study
Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Spontaneous avirulent mutant from Xc11, Apr Xc11 blaXCC-1 mutant constructed by inserting a Tcr cartridge into the SmaI site of the blaXCC-1 gene, Tcr/Aps Xc11 ampR1 mutant constructed by inserting a Tcr cartridge into the SalI site of the ampR1 gene, Tcr/Aps Xc11 blaXCC-1 mutant complemented with cloned blaXCC-1 gene, Tcr/Apr/Kmr Xc11 ampR1 mutant complemented with cloned ampR1 gene, Tcr/Apr/Kmr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Aps Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr Wild-type strain isolated in Taiwan, Apr
Tseng lab collectiona Tseng lab collection Tseng lab collection Yang and Tseng (33) Yang and Tseng (33) Yang and Tseng (33) Weng et al. (29)
Wild-type strain isolated in Taiwan, Apr European strain, Aps Wild-type strain isolated in Taiwan, Apr
Tseng lab collection Ojanen et al. (20) L. T. Wu, China Medical University
This study Weng et al. (29) This study Tseng Tseng Tseng Tseng Tseng Tseng Tseng
lab lab lab lab lab lab lab
collection collection collection collection collection collection collection
Yi-Hsiung Tseng’s laboratory collection.
pFJ-ampR1 was constructed by cloning the 1.3-kb PstI-XbaI fragment from the pFY13-9BSRX insert, containing the entire ampR1 gene, into the broad-hostrange vector pFJ9 (7). Plasmid pOKSS was a derivative of pOK12 carrying the 1.0-kb SmaI-SstI fragment from the pBKE insert. Plasmid pBX contained the 500-bp PstI-PvuII DNA fragment carrying the blaXCC-1-ampR1 IG (from the pFJ-ampR1 insert) with the blaXCC-1 promoter being situated in the same orientation as and upstream of the luxA and luxB reporter genes in promoterprobing vector pMY3 (31). Plasmid pRX contained the same insert as that of pBX except that the orientation was opposite, thus placing the reporter genes under the control of the ampR1 promoter. Construction of the ampR1 mutant, Xc11RM. Strain Xc11RM was constructed by insertional mutation. The 2.0-kb tetracycline resistance (Tcr) cartridge from mini-Tn5Tc (9) was inserted into the SalI site within ampR1 of the pCH1 insert. The resultant plasmid with the interrupted ampR1 gene was then electroporated into strain Xc11 to replace the chromosomal wild-type version. One of the mutants (Xc11RM), resistant to tetracycline and sensitive to ampicillin, thus obtained was verified to have undergone a double crossover by Southern hybridization. -Lactamase assays. The activity of -lactamase was measured spectrophotometrically by monitoring the hydrolysis of nitrocefin (Calbiochem-Novabiochem Corp.) at room temperature as described previously (11), using the cell extracts prepared by sonication as the enzyme sources. One unit of enzyme was defined as the amount required to hydrolyze 1 nmol of nitrocefin per min. The protein contents were determined by the method of Bradford (4) with bovine serum albumin as the standard.
IEF analysis. Isoelectric focusing (IEF) analysis of -lactamase in the crude extracts prepared from cultures of the Xanthomonas strains grown overnight was performed in PhastSystem (Amersham Biosciences) with precast gels (pI 3 to 9). The bands were visualized by overlaying the IEF gels with filter paper soaked in a 2.0-ml mixture prepared by combining 1.9 ml of 100 mM sodium phosphate buffer (pH 7.0) with 0.1 ml of nitrocefin (10 mg/ml in dimethyl sulfoxide). Antibiotic sensitivity of Xanthomonas. The MICs of different -lactam antibiotics for the Xanthomonas strains were determined by using Etest strips (AB Biodisk, Solna, Sweden) or by the microdilution method recommended by the National Committee for Clinical Laboratory Standards, using -lactams purchased from Sigma (St. Louis, Mo.). E. coli ATCC 25922 was used as the reference strain. Primer extension. A oligonucleotide primer with a sequence complementary to positions 80 to 104 downstream of the ampR1 start codon (5⬘-GCCTGGCT GACGCACAGCTCGCCGG-3⬘; designated primer R2) was radioactively labeled as previously described (30). Total RNA was isolated from cells of strain Xc11 grown to mid-exponential phase by the Qiagen RNA extraction system (Qiagen, Valencia, Calif.) according to the manufacturer’s instructions. The reverse transcription was performed using the labeled primer and the extracted RNA (30 g) as the template. The same primer was used for DNA sequencing on the insert of pOKSS (Fig. 1) at the same time to identify the positions of the transcriptional start sites. DNA methods. Preparation of plasmid and chromosomal DNA, restriction enzyme digestion, Southern blotting, and transformation of E. coli DH5␣ were performed by standard procedures (22). DNA sequences on both strands were
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FIG. 1. Inserts of the plasmids carrying blaXCC-1 and/or ampR1 from X. campestris pv. campestris strain 11. The 4.0-kb Sau3A1 fragment in pCH1, the 2.5-kb StuI-XbaI fragment in pFY13-9BSRX, the 1.7-kb EcoRI-SstI fragment in pBKE, the 0.5-kb PstI-SstI fragment in pBKX, the 1.0-kb SmaI-SstI fragment in pOKSS, the 1.3-kb PstI-XbaI fragment in pFJ-ampR1, the 0.5-kb PstI-PvuII fragment in pBX, and the same fragment in the opposite orientation in pRX are shown. The underlined restriction sites are from the multiple cloning sites of the vectors.
determined by the dideoxy chain termination method (24). Plasmid was delivered to X. campestris by electroporation (28). Luciferase activity determination. Luciferase activity was assayed by measuring bioluminescence (in volts per milliliter) as described previously (15) by monitoring cell growth (from an optical density at 660 nm of approximately 0.05 to 2.0). The maximal values obtained from each culture were used for comparison. Computer analysis. The BLAST program was used to search the National Center for Biotechnology Information database for AmpR homologues. The dendrogram was constructed by using the Clustal X program. Nucleotide sequence accession number. The nucleotide sequence of 1,046 bp (from the blaXcc-1-ampR1 IG to the XmnI site) determined here has been deposited in GenBank under accession number AY165954.
RESULTS AND DISCUSSION Sequence analysis of X. campestris pv. campestris ampR. In our previous study of the X. campestris strain Xc11 -lactamase gene (blaXCC-1), the sequence of the upstream region including the N-terminal 41 codons of the regulatory ampR1 gene was also delineated (29). In this study, the entire ampR1 gene including the region further upstream (Fig. 1) was sequenced. The relative positions of the blaXCC-1 and ampR1 genes, contained within the 2.5-kb EcoRI-XmnI fragment of the pFY139BSRX insert, are shown in Fig. 1. Running opposite to blaXCC-1, the Xc11 ampR1 gene encoded a polypeptide of 289
amino acid (aa) residues with a calculated molecular size of 31 kDa, named AmpR1. Similar to the situations of other X. campestris chromosomal genes, the ampR1 gene had a G⫹C content of 70%, and G and C were strongly favored at the third positions of the codons (79%). A region having the potential to form a stem-loop structure that resembles the transcriptional terminator was found 9 bp downstream from the ampR1 termination codon, spanning 27 nucleotides (nt) with a calculated ⌬G of ⫺19.4 kcal/mol. There was no string of T residues characteristic of typical Rho-independent transcriptional terminators following the stem-loop structure. Sequence comparisons of XCC and AmpR proteins. A computer search revealed that the X. campestris pv. campestris ATCC 33913 genome had four -lactamase genes (synonym XCC3039, XCC2636, XCC2873, and XCC3197, encoding enzymes XCC-2, XCC-3, XCC-4, and XCC-5, respectively) and two ampR genes (synonym XCC3040 and XCC3781) (8). XCC3039 and XCC3040 were adjacent and running divergently, which suggests that they are cognate structural and regulatory genes. Sequence comparisons revealed that XCC-1 (295 aa) (29) and AmpR1 had 99 and 94% identity with XCC-2 (296 aa) and XCC3040 (291 aa), respectively. Low degrees of identity (29 to 35%) were shared between the amino acid
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FIG. 2. Dendrogram of AmpR proteins based on the sequence similarity between the AmpR1 protein and 17 other sequences with the highest identity to AmpR1. The percent identity with AmpR1 is shown in parentheses.
sequences of the X. campestris paralogues, XCC-1 versus the other XCC proteins and AmpR1 versus XCC3781. These observations suggest that strain Xc11, the same pathovar as ATCC 33913, may also possess multiple bla genes. However, since mutation in blaXCC-1 renders the strain sensitive to -lactam antibiotics (29), the other blaXCC genes may not be required for resistance. One homologue to each of these Bla proteins was found in Xanthomonas axonopodis pv. citri 306 genome, XCC-6, XCC-7, XCC-8, and XCC-9 (8), which corresponded to XCC-2, XCC-3, XCC-4, and XCC-5 with 81, 86, 95, and 84% identities, respectively. The AmpR1 protein also shared high degrees of identity with the X. axonopodis pv. citri 306 AmpR1 protein (synonym XAC3163; 81%), Pseudomonas aeruginosa AmpR (58.2%), and Burkholderia cepacia LysR (54.2%) and shared moderate degrees of identity (more than 45%) with several other bacterial AmpR proteins. However, although moderate to high degrees of identity were shared between these AmpR proteins, phylogenetic analysis revealed that the three AmpR1 proteins from Xanthomonas clustered independently and that the two
AmpR2 proteins formed still another cluster, which was even further apart from the other clusters (Fig. 2). The low homology between AmpR1 and AmpR2 of the Xanthomonas strains suggests that they might be derived from different ancestors. AmpR proteins are members of the LysR family of transcriptional regulators that possess a helix-turn-helix motif at their N terminus required for DNA binding (25). With computer analysis using NPS@, developed by PBIL Lyon-Gierland (http://pbil.ibcp.fr/html/), a helix-turn-helix motif was also found at the N-terminal region in AmpR1 and XCC3040 (with the same sequence at the same position, NLTRAAGELCVSQ AALSHQIK, aa 22 to 42) and XCC3781 (NLSRAAAAMHL TVSALSHQVR, aa 20 to 40). The blaXCC-1-ampR1 IG contains partially overlapped promoters with low AⴙT content. The bla-ampR IGs from nine strains of gram-negative bacteria were aligned with those of Xc11 and ATCC 33913 (Fig. 3). The blaXCC-1-ampR1 IG, with identical nucleotide sequence to and the same length (100 bp) as that of XCC3039-XCC3040, was shorter than the IG from other bacteria, which range from 127 to 151 bp (Fig. 3). Struc-
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FIG. 3. Alignment of the bla-ampR IGs from nine bacterial strains with that of X. campestris pv. campestris strains Xc11 and ATCC 33913, which have the same length and sequence. Start codons and promoters (⫺10 and ⫺35 sequences) are shaded and indicated below the sequences, and the promoter of blaXCC-1 is underlined. Conserved regions 1 and 2 predicted for AmpR binding are indicated above the sequences, and the putative LysR motif within region 1 is underlined. ⫹1 is the transcription start site determined for the ampR1 promoter.
tural features typical of the bla-ampR IG present in other bacteria were also found in the blaXCC-1-ampR1 IG, including the conserved regions 1 and 2 predicted for AmpR binding and the LysR motif AN11T (Fig. 3). These IGs are commonly AT-rich and possess promoters with ⫺10 and ⫺35 sequences typical of E. coli 70 promoters. However, the A⫹T content in the X. campestris blaXCC-1-ampR1 IG was low (37%), and no regions conserved to the E. coli 70 promoters were found (Fig. 3). This latter finding is different from our previous observations that most of the X. campestris promoters are rich in A⫹T,
in contrast to the overall low A⫹T content of the chromosome (31). Sequence alignments suggested that the sequences 5⬘-AAT GTT-3⬘ and 5⬘-CGGCGC-3⬘ located 36 and 58 nt upstream of the ampR initiation codon with a 16-nt spacer between the two sequences are the ⫺10 and ⫺35 sequences, respectively, of the ampR1 promoter (Fig. 3). The same alignment suggested that the blaXCC-1 promoter had 3⬘-AGTATC-5⬘ and 3⬘-CGACC A-5⬘ (on the opposite strand) located 27 and 50 nt upstream of the blaXCC-1 initiation codon as the ⫺10 and ⫺35 sequences, respectively, suggesting that the blaXCC-1 and ampR1 promoter regions overlapped by 18 nt (Fig. 3). Primer extension was performed to determine the transcription initiation site of ampR1. Extension reaction using primer R2 (5⬘-GCCTGGCTGACGCACAGCTCGCCGG-3⬘), complementary to nt 80 to 104 relative to the ampR initiation codon, yielded one extension product initiating with a G base
TABLE 2. MICs of -lactam antibiotics for various strains testeda -Lactam
FIG. 4. Determination of the transcriptional start site of ampR1. The product of primer extension (arrow) and the base complementary to the 5⬘ end of the transcript (asterisk) are indicated.
Ampicillin Oxacillin Piperacillin-tazobactam Carbenicillin Cefoxitin Cefotaxime Imipenem
MIC (g/ml) Xc11
Xc11RM
Xc11RM(pFJ-ampR1)
⬎256 ⬎256 256 256 128 32 4
4 64 24 16 3 8 0.25
⬎256 ⬎256 256 512 128 32 4
a All tests were performed with Etest strips, except that carbenicillin and cefotaxime were tested by the microdilution method.
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TABLE 3. -Lactamase activities for various strains tested -Lactamase activitya Strain
Xc11 Xc11RM Xc11RM(pFJ-ampR1) Xc17 Xc6 Xv64 Xv3240 Xcm38 Sm6
Gene
blaXCC-1 blaXCC-1 blaXCC-1 blaXCC-10 blaXCC-11 blaXAV-1 Unknownb —c blaSM-1
Without cefotaxime
With cefotaxime
3,670 ⫾ 80 7⫾2 3,200 ⫾ 44 4,190 ⫾ 30 3,590 ⫾ 72 10,000 ⫾ 88 38 ⫾ 18 28 ⫾ 7 1,590 ⫾ 71
3,550 ⫾ 144 ND 3,120 ⫾ 25 4,150 ⫾ 78 3,320 ⫾ 89 9,960 ⫾ 58 ND ND 5,890 ⫾ 147
a The specific activity of the -lactamase is defined as nanomoles of nitrocefin hydrolyzed per minute per milligram of protein and the mean ⫾ standard deviation is shown for each strain. Cells were grown without cefotaxime or with cefotaxime at a final concentration of 8 g/ml. ND, not determined. b Presence of the gene not determined. c —, no blaXCC-1 homologue detected by Southern hybridization.
that was 26 nt upstream from the ampR initiation codon and 9 nt downstream from the predicted ⫺10 box (Fig. 4). Expression of the blaXCC-1 gene is constitutive and requires the function of AmpR1. MICs ranged from 4 (imipenem) to more than 256 g/ml (ampicillin, oxacillin, piperacillin-tazobactam, and carbenicillin) against the Xc11 strain, while MICs decreased drastically against the Xc11RM strain, with reductions ranging from 4 to 64 times lower than the wild-type levels of resistance (Table 2). The wild-type levels of resistance to -lactams were restored in the mutant containing the cloned ampR1 gene, Xc11RM(pFJ-ampR1) (Table 2). Notably, the data indicated reduced susceptibility to imipenem and resistance to cefoxitin similar to Xc11. These are different from the situations with other class A -lactamases. The reason remained to be studied. However, since XCC-1 shares only 51.5% sequence identity with the S. maltophilia L2 -lactamase (29), the most closely related class A enzyme, XCC-1 may have varied hydrolytic properties for these substrates. To test whether susceptibility to -lactams caused by an ampR1 mutation correlated with the loss of the responsible enzymatic activity and whether expression of blaXCC-1 can be induced, -lactamase was assayed. Cultures were grown to exponential phase (ca. 0.5 U of optical density at 550 nm) without -lactams, divided into two parts, and grown for 2 h with or without cefotaxime (8 g/ml). In strain Xc11RM, -lactamase activity was as low as 7 U/mg of protein. Complementation of Xc11RM with pFJ-ampR1 restored the enzyme activity (3,200 ⫾ 44 U/mg), which was comparable to that in the wild-type Xc11 (3,666 ⫾ 80 U/mg) (Table 3). These results, demonstrating the activator function of AmpR, indicate that a mutation in the ampR1 gene caused a simultaneous loss of the -lactamase activity, which in turn resulted in susceptibility to -lactam antibiotics. The positive control of AmpR1 over blaXCC-1 expression is thus similar to the ampR-ampC system in Enterobacteriaceae. With cefotaxime, the enzyme levels in strains Xc11 (3,547 ⫾ 144 U/mg) and Xc11RM(pFJ-ampR1) (3,122 ⫾ 25 U/mg) were about the same as those without cefotaxime (Table 3), suggesting that the expression of blaXCC-1 gene is constitutive and cannot be further induced by cefotaxime. In the mutant
cells (Xc11RM), while the cells were not able to grow in the presence of cefotaxime, no significant levels of -lactamase were detected without cefotaxime either. In the parallel experiments using Serratia marcescens 6 (strain Sm6) as a positive control, the addition of 8 g of cefotaxime per ml caused a fourfold increase in the -lactamase activity (5,887 ⫾ 147 versus 1,591 ⫾ 71), suggesting that induction would have been observed if it occurred in Xanthomonas. In Enterobacter, Citrobacter, and Pseudomonas, both hyperinducible and highlevel constitutive -lactamase production are due to mutations in ampD (5, 13). Because an ampD homologue is present in X. campestris pv. campestris 33913 genome (8), studies are in progress to determine whether constitutive expression of blaXCC-1 is caused by a mutation in ampD. Several other Xanthomonas strains were also assayed for -lactamase levels, including X. axonopodis pv. vesicatoria 64 (strain Xv64), X. axonopodis pv. vesicatoria 3240 (strain Xv3240), X. campestris pv. campestris 6 (strain Xc6), X. campestris pv. campestris 17 (strain Xc17), and X. campestris pv. mangiferaeindcae 38 (strain Xcm38) (Table 3). As expected, the ampicillin-sensitive strains Xv3240 and Xcm38 did not produce significant levels of the enzyme. In contrast, the enzyme levels in strains Xc17 and Xc6 were similar to that in Xc11, whereas those in Xv64 were about threefold higher than that in Xc11. The addition of cefotaxime did not affect the levels of -lactamase in these strains, again suggesting that the enzyme is constitutively expressed. Since the -lactamase-producing strains were all wild-type bacteria, it appears that constitutive expression of -lactamase is intrinsic in Xanthomonas. Since Xanthomonas is a large genus and inhabits soil, infected plants, plant debris, and asymptomatic plants near acutely infected plants, constitutive expression of -lactamase by these bacteria raises environmental concerns regarding dissemination of the resistance gene. To obtain -lactamase profiles, IEF was performed with crude extracts prepared from strains including Xc11, Xc11RM, Xc11RM(pFJ-ampR1), 11bla::Tc, 11bla::Tc(pRKSE), Xc6, Xc17, Xv64, and Xcm38. A single band was localized near pI 8.2, similar to that predicted for XCC-1 (29), in all strains grown with or without cefotaxime, except in strains 11bla::Tc, Xcm38, and Xc11RM which did not display significant levels of -lactamase (data not shown). These data indicated the existence of only one species of -lactamase in each of these strains. Notably, Xv64 displayed a band with much stronger intensity than those of the other strains (data not shown), consistent with the data that Xv64 exhibited threefold-higher levels of -lactamase than Xc11. The ampR1 promoter is not active in E. coli. Because the A⫹T content of the blaXCC-1-ampR1 IG was low, which differs from the typical 70 promoters, we were curious about whether the promoters were able to express in E. coli. For the test, plasmid pFY13-9BSRX (Fig. 1) carrying the entire ampR1 and blaXCC-1 genes was introduced into E. coli DH5␣. The transformant, E. coli(pFY13-9BSRX), was resistant to kanamycin but unable to grow in L agar plate containing ampicillin, indicating that while the plasmid-encoded kanamycin resistance was functional, -lactamase was not produced by the cloned blaXCC-1 gene in E. coli. Results of enzyme assays confirmed that no significant levels of -lactamase activity were present. Reporter plasmids pBX and pRX with transcriptional fu-
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sions (Fig. 1) were used to assay for the blaXCC-1 and ampR1 promoter activities, respectively. No significant levels of luciferase activity were detected in either of the constructs, indicating that both blaXCC-1 and ampR1 promoters were not active. The observation that ampR1 promoter is not expressed is consistent with our previous report that most of the Xanthomonas promoters are not expressed in E. coli (31). Since expression of the blaXCC-1 promoter requires AmpR1, no expression of Xc11 blaXCC-1-ampR1 system in E. coli is thus explained. Distribution of the ampR gene in xanthomonads. We have previously shown that 13 Xanthomonas strains tested, except Xcm38, are all resistant to ampicillin and contain a bla gene detectable by Southern hybridization (29). In this study, the labeled 240-bp PvuII-SalI DNA fragment within the ampR1 gene was used as a probe for Southern hybridization with the EcoRI-digested chromosomal DNA from these Xanthomonas strains. We detected a 3.8-kb fragment in X. campestris pathovars campestris (Xc2, Xc6, Xc11, Xc11A, Xc17, and Xc85), begoniae (Xcb59), dieffenbachiae (Xcd65), and phaseoli (Xcp73), a 10-kb fragment in X. axonopodis pathovars citri (Xac60), glycines (Xcg69), and vesicatoria (Xv64), and a 5.5-kb fragment in X. oryzae pv. oryzae (Xo21), but none in the ampicillin-sensitive Xcm38 (data not shown). These observations together indicate that each of the ampicillin-resistant Xanthomonas strains contains genes homologous to blaXCC-1 and ampR1.
10. 11.
12. 13. 14.
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16. 17.
18. 19.
ACKNOWLEDGMENTS This study was supported in part by grant NSC-90-2311-B005-025 from the National Science Council and grant 91-B-FA05-14AA91002A of the Program for Promoting Academic Excellence of Universities from the Ministry of Education of the Republic of China.
20. 21.
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