Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
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Abstract |
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Introduction |
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Some strains of Aeromonas spp. produce multiple ß- lactamases, as demonstrated by biochemical and genetic analysis.69 Analysis of an A. veronii bv. sobria mutant with increased resistance to ß-lactams (strain 163aM) revealed the co-ordinate overexpression of three ß-lactamases.7 Another study reported similar findings for the Aeromonas jandaei mutant AER14M, where increased expression of at least two ß-lactamases was observed.10 The genes for the three ß-lactamases of A. veronii bv. sobria have been cloned and sequenced and designated ampS, cepS and imiS.11,12 AmpS is a class 2d penicillinase, CepS is a class 1 cephalosporinase and ImiS is a class 3 metallo-ß-lactamase.1113 Each ß-lactamase has a very narrow hydrolytic spectrum of activity, with CepS hydrolysing only cephalosporins, ImiS active mainly against carbapenems and AmpS hydrolysing mainly penicillins.11,12 Two ß-lactamase genes have been sequenced from A. jandaei AER14, namely asbA1 and OXA-12 (asbB1),10 but only the metallo-ß-lactamase gene, cphA, has been cloned and sequenced from A. hydrophila.14 We have shown that A. hydrophila isolates have homologues of ampS and cepS, as judged by hybridization studies,9 but until now, no information has been presented concerning the nucleotide sequence of these genes.
In Gram-negative bacteria such as Citrobacter freundii, inducible expression of the ß-lactamase gene, ampC, is regulated by the product of the linked gene, ampR, which encodes a transcription factor of the LysR family.15 This type of regulator usually works in conjunction with a diffusible activator ligand, which, for AmpR, is believed to be 1,6-anhydro-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid-D-alanine-D-alanine,16,17 a product of peptidoglycan breakdown that is released more rapidly when cells are exposed to ß-lactam drugs.18,19 This mechanism of controlling ß-lactamase expression is also found in other members of the Enterobacteriaceae, as well as in Pseudomonas aeruginosa.20 In all these organisms, however, there is expression of a single ß-lactamase (AmpC), which is in contrast to the co-ordinated expression of three enzymes in Aeromonas spp.
In Escherichia coli, the Cre regulon21 has been implicated in the expression of ß-lactamase genes cloned from A. jandaei AER14; given that undefined E. coli cre mutants overproduce AER14 ß-lactamases.10 In addition, an A. jandaei gene, blrA, encoding a CreB homologue, was found to enhance the expression of cloned AER14 ß-lactamase genes when co-expressed in E. coli.22 However, the manner in which the Cre regulon influences ß-lactamase expression, and therefore the mechanism of ß-lactamase induction in Aeromonas spp., remains to be resolved.
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Materials and methods |
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A. hydrophila T429125 was a clinical isolate from the Royal Hobart Hospital, Tasmania, Australia. E. coli strains used were DH5 [supE44,
lacU169 (
80 lacZ
M15), hsdR17, recA1, endA1, gyrA96, relA1, thi, phoR68, cre+];23 UB5252 (thi, rfaG::Tn5);24 and RB2181 (supE44, lacY, tonA, thi, leu, str, phoR68,
serB-thr).25
Materials
Unless otherwise stated, media used were nutrient broth and nutrient agar (Oxoid plc, Basingstoke, UK). Meropenem was from Zeneca Pharmaceuticals, Macclesfield, UK. All other reagents were from Sigma or BDH, both of Poole, Dorset, UK.
Induction and preparation of ß-lactamases
Bacterial strains were cultured overnight in nutrient broth with shaking at 37°C. A 1 in 20 dilution of each culture was made into 50 mL of fresh broth and the culture was shaken at 37°C until a turbidity (OD420) of 0.8 was reached. Inducer (cefoxitin) was then added to the culture in 50 mL of fresh pre-warmed broth, to give a final concentration of 10 mg/L, and incubation was continued for 2 h. To assay ß-lactamase activity, bacteria from 10 mL of culture were harvested by centrifugation (10 min, 4°C, 3500g) and washed twice in 10 mL ice-cold extraction buffer {50 mM MOPS [3-(N-morpholino)propane sulphonic acid], pH 7.0}. Following resuspension of the pellet in 1 mL of extraction buffer, the cells were disrupted in a Ribolyser (Hybaid, Teddington, UK) in tubes containing silica beads (Hybaid Blue matrix), with a single 30 s burst (amplitude 6). Cell debris and silica beads were pelleted by centrifugation (10 min, 4°C, 15000g) and the supernatant was transferred to a clean tube and used directly as a source of ß-lactamase.
ß-Lactamase assays
Hydrolysis of ß-lactam antibiotics was examined by spectrophotometric analysis using a Pharmacia LKB Ultraspec III (Pharmacia, St Albans, UK) in 1 cm light-path cuvettes with readings recorded every 2 s for 3 min at the wavelength of optimal absorbance of the ß-lactam ring of each drug (i.e. 233, 265, 299 and 250 nm for ampicillin, cephaloridine, meropenem and oxacillin, respectively). Antibiotic solutions (100 µM) were prepared in 50 mM MOPS, pH 7.0. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturer's instructions. Extinction coefficients used were 656, 7374, 2500 and 240 absorbance units/M/cm for ampicillin, cephaloridine, meropenem and oxacillin, respectively. One unit of ß-lactamase activity was defined as that required to hydrolyse 1 nmol of substrate per minute at 25°C. Specific activity was defined as the number of units of ß-lactamase per milligram of protein in the cell extract.
Cloning of ß-lactam-resistance genes
Chromosomal DNA was prepared from A. hydrophila T429125 as described previously.26 The DNA was digested with SalI or BamHI, ligated into appropriately digested pSU1827 or pK18,28 respectively, and used to transform E. coli strains DH523 or UB525224 by electrotransformation using a Gene Pulser (Bio-Rad) set at 2.5 V, 25 mF and 200
. Transformed bacteria were selected on medium containing kanamycin or chloramphenicol (both at 30 mg/L), as appropriate for the cloning vector. To recover the cephalosporinase and penicillinase genes, respectively, transformants were selected also for resistance to cephaloridine or ampicillin (both at 20 mg/L). Resistant transformants were recovered, plasmid DNA was isolated and the insert was sequenced using an ABI PRISM 377 automated DNA sequencer. Sequences were determined on both strands using a custom primer-walking strategy.
Production of a DH5 creB insertion mutant
The creB gene from DH5 was amplified by PCR, by the method described previously,29 using primers based on its published sequence30 (i.e. 5'-GTCTGGCGGAAGATACCT-3' and 5'-CATTACAGGCCCCTCAGG-3'). The creB gene contains a unique KpnI restriction site,30 which was exploited to disrupt the gene. To do so, the aph type I kanamycin resistance gene (including its promoter) was amplified from plasmid pK1828 by PCR using primers that inserted a KpnI restriction site on either side of the product (i.e. 5'-GCCAGCTGGGGTACCCTCTGGTAAG-3' and 5'-GGCGGCGGTGGTACCGAAATCTCGTG-3'). Both creB and aph PCR amplicons were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and then digested with KpnI.26 The resultant fragments were purified as above, mixed and ligated.26 PCR with the creB primer set was repeated using the ligation reaction as a template, and produced two amplicons: one, an intact creB gene, and the other, the creB gene with an aph insertion. The amplicons were separated by gel electrophoresis and the larger, with the aph insertion, was gel purified using a Qiagen QIAquick gel extraction kit and used as a template for a further round of creB PCR (primers as before) to recover large amounts of the creB::aph amplicon. The amplicon was purified as before and 1 µg was used to electrotransform c.108 DH5
cells.26 Five colonies resistant to kanamycin 30 mg/L were recovered where the creB gene had been replaced by creB::aph. The aph insertion into the chromosomal copy of creB was confirmed by PCR. One of these mutants was thus designated DH5
(creB::KmR)
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Results |
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A. hydrophila T429125 was challenged with cefoxitin 10 mg/L for 2 h to induce the production of ß-lactamases and cell extracts were examined for their ability to hydrolyse ampicillin, cephaloridine and meropenem (Table I). In the uninduced state, the production of ß-lactamase was minimal and activity was detectable only against ampicillin. Following cefoxitin challenge, ß-lactamase activities against all three substrates increased significantly. Given our prior knowledge of Aeromonas spp. ß-lactamases,7,9 the fact that the extracts of induced cells hydrolysed all three ß-lactams (Table I
) indicates that the expression of the three ß-lactamases in T429125 is co-ordinately inducible.
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A SalI gene bank was constructed in vector pSU18 from genomic DNA of A. hydrophila T429125 and used to transform E. coli DH5. Transformants were selected on nutrient agar containing cephaloridine 20 mg/L; each contained a recombinant plasmid with a 5.8 kb insert. One such plasmid was designated pUB5962.
Several attempts were made to recover ampicillin-resistant clones of E. coli DH5 after transformation with a pK18-derived A. hydrophila T429125 BamHI gene bank, but none was successful. Accordingly, E. coli UB5252 was used as the recovery strain. This strain has mutations that reduce the levels of OmpF and OmpC porins in the outer membrane. This reduction in porins diminishes the rate of ß-lactam passage across the outer membrane, resulting in an increased resistance to ß-lactams for a given level of ß-lactamase.24 Using UB5252, therefore, increases greatly the chance of recovering ß-lactamase genes that are expressed poorly in standard laboratory E. coli strains such as DH5
.24 When UB5252 was transformed with the A. hydrophila T429125 BamHI gene bank, transformants were recovered on medium containing ampicillin 20 mg/L. All of the transformants tested carried a plasmid containing a 4.4 kb insert and one such plasmid was designated pUB5972.
The chromosomal inserts from pUB5962 and pUB5972 were used as probes to hybridize with A. hydrophila T429125 genomic DNA. The results were positive in both cases, confirming the genes origin (data not shown).
Sequence analysis of cepH
Sequencing of cepH, carried on plasmid pUB5962, was initiated using primers designed from the cepS sequence,11 given the high degree of similarity of the two genes (as indicated by Southern hybridization; data not shown). Using a primer-walking strategy, the entire sequence of cepH was determined (Figure 1; EMBL accession number AJ276030). The cepH gene extends for 1149 nucleotides, encoding a peptide of 382 amino acids. A credible ribosome-binding site is located eight nucleotides upstream of the initiation codon; 51 nucleotides downstream of the gene there is an inverted repeat followed by a poly(T) run, which is likely to be a Rho-independent transcription terminator.31 No obvious promoter with significant homology to the
70 promoter of E. coli has been identified for cepH (Figure 1
).32 A 23 amino acid secretion leader peptide33 in CepH is defined by alanine residues at positions 23 and 24. The mature 359 amino acid protein has a predicted molecular weight of 38.3 kDa and a predicted pI of 6.6, while the amino acid motif characteristic of serine active-site ß-lactamases, SVSK, is located at positions 6265 in the mature protein (Figure 1
). The predicted amino acid sequence of CepH shows highest similarities with the class 1 cephalosporinases of A. jandaei AER 14 (AsbA1, 86% identical)10 and A. veronii bv. sobria 163a (CepS, 78% identical).11 The enzyme is also clearly related to the AmpC ß-lactamase (42% identical) from E. coli.34
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A primer-walking strategy was also used to sequence ampH, with sequencing initiated using primers based on the ampS sequence.11 The ß-lactamase gene extends for 795 nucleotides (Figure 2) (EMBL accession number AJ276031) and encodes a peptide of 264 amino acids. It is preceded by a credible ribosome-binding site and there is a possible Rho-independent transcription terminator seven nucleotides beyond the end of the gene.31 Putative 10 and 35 sequences were identified upstream of the gene; however, a separation of 20 nucleotides between the two sequences (Figure 2
), compared with the optimum of 16 or 17 for the E. coli
70 promoter,32 casts doubt on their authenticity. The primary translation product has a strong hydrophobic N terminus, consistent with a secretion leader of 22 amino acids. This would be cleaved from the preprotein between two alanines at positions 22 and 23 (Figure 2
).33 The mature 242 amino acid protein, with a predicted molecular weight of 26.9 kDa, has a predicted pI of 8.0. Amino acids 5154 of the mature enzyme contain the serine active-site motif, STFK (Figure 2
). A comparison of the predicted amino acid sequence of AmpH with those of other ß-lactamases revealed identity to class 2d oxacillinase enzymes OXA-1 (42% identity)35 and BLAD (64% identity),36 with greatest identity shown to AsbB1 (82%) from A. jandaei10 and AmpS (85%) from A. veronii bv. sobria.11
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It has been reported that undefined mutations at the E. coli cre locus can enhance expression of cloned A. jandaei ß-lactamase genes.10 An E. coli creABCD deletion mutant, strain RB2181,25 was therefore used to assess whether the wild-type Cre regulator affects expression of the cloned cepH and ampH genes. The plasmids pUB5962 and pUB5972 were separately transformed into E. coli DH5 (cre+) and RB2181; transformants were selected with chloramphenicol or kanamycin (both at 30 mg/L), as appropriate. Both CepH and AmpH were expressed more in RB2181 than in DH5
, as determined by relative rates of hydrolysis of cephaloridine and oxacillin, respectively (Table II
).
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An additional factor regulates the expression of AmpH and CepH in RB2181
It was interesting to note that, in RB2181, the expression of CepH was c.11 times that in DH5, but the expression of AmpH was only three times that in DH5
. To investigate the possibility that an additional factor, carried on the insert of pUB5962, was responsible for the additional enhancement of cepH expression, DH5
and RB2181 derivatives containing both pUB5962 (cepH) and pUB5972 (ampH) were constructed. AmpH activities in cell extracts were determined from the rate of oxacillin hydrolysis (CepH does not hydrolyse this compound) (Table II
). The production of AmpH by RB2181:(ampH):(cepH) was 12 times that by DH5
:(ampH):(cepH) and some four-fold more than that by RB2181:(ampH) (Table II
). Hence, in RB2181, expression of ampH is enhanced significantly by the presence of pUB5962.
To investigate the nature of the trans-activating factor encoded on the insert of pUB5692, responsible for the increase in cepH and ampH expression in RB2181, the entire sequence of the pUB5962 insert was determined (EMBL accession number AJ 276030). No candidate gene encoding an obvious transcriptional regulator could be identified. Four putative open reading frames, in addition to cepH, are found on the insert (Figure 3). ORF2 was truncated during the cloning procedure; neither it nor ORF1 shows any homology to other gene sequences deposited on the usual databases. The third putative ORF is predicted to encode a 499 amino acid membrane protein, with 75% amino acid identity to a putative E. coli protein, YhiP.34 From its amino acid sequence, E. coli YhiP may be an outer membrane dipeptide or tripeptide transport protein, but nothing is known about this protein or its production.34 The fourth ORF could encode a 142 amino acid cytoplasmic protein with 64% amino acid identity to a putative E. coli protein, YqgF,34 of which, again, nothing is known.
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Discussion |
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We have shown previously that A. veronii bv. sobria produces three chromosomal ß-lactamases.7 Expression of the three enzymes is normally repressed but is induced by ß-lactams, and genetic derepression results in high-level expression of all three.7 Similar observations have been made for A. jandaei.10
In this report, we have shown that A. hydrophila strain T429125 also produces three ß-lactamases (Table I) which are co-ordinately induced in response to ß-lactam challenge. The metallo-ß-lactamase gene from A. hydrophila, cphA, has been cloned and sequenced previously.14 There is an almost identical enzyme in A. hydrophila T429125, as determined by PCR analysis and sequencing of the resulting amplicon (data not shown). Here we report the sequences of the cepH and ampH serine ß-lactamase genes from this species. The predicted amino acid sequences and hydrolytic activities of the enzymes reveal that CepH is a class 1, AmpC-like cephalosporinase, while AmpH is a class 2d oxacillinase,13 as are the A. veronii bv. sobria and A. jandaei equivalents.10,11 A more thorough examination of the biochemical properties of these newly discovered enzymes will await further information concerning the structures and mechanisms of OXA and AmpC-type ß-lactamases in general.
Regulation of CepH and AmpH expression in E. coli
Our current understanding of the induction of AmpC-type ß-lactamase is based mainly on studies of the ß-lactamases of C. freundii and Enterobacter cloacae, where the ampC gene is linked to a divergently transcribed regulator gene, ampR, with a separating intercistronic region of 80150 bp.15,37 Analysis of DNA directly upstream from ampH, cepH (Figures 1 and 2) and cphA14 has not revealed an open reading frame related to ampR.
Rasmussen and colleagues10 have reported that undefined mutations in the E. coli Cre regulon increased expression of the A. jandaei ß-lactamases, which were otherwise expressed poorly in E. coli. Our experiments indicate that expression of the ampH and cepH genes is significantly greater in E. coli when the cre genes are absent or inactive (Table II). These findings suggest that a component of the Cre regulon represses transcription of the A. hydrophila ß-lactamase genes. A likely candidate for this is the putative transcription factor, CreB,21 and specific disruption of creB has added strong evidence for this hypothesis (Table II
).
Removal of the Cre repressor revealed that the A. hydrophila chromosomal insert of plasmid pUB5962 encodes a function that can trans-activate the expression of ampH, increasing it to approximately four times that seen in the cre mutant alone (Table II
). We could not identify this element from the sequence of the insert (Figure 3
). In particular, there are no genes on the insert that show homology to those of known classes of transcriptional regulators or to RNA polymerase
factors.34
It is likely that, as well as performing the repressive function demonstrated here, the Cre two-component system (TCS) can activate the expression of Aeromonas spp. ß- lactamases in some situations. Many other TCSs act as repressor/activator switches for the transcription of target genes, for example the PhoBR TCS, which is highly homologous to CreBC.21 We think it significant that introduction of blrA, an A. jandaei homologue of creB, into a cre+ E. coli background activates the expression of cloned A. jandaei ß-lactamases, but that such an effect was only observed when blrA was cloned from an A. jandaei ß-lactamase-hyperproducing mutant.22 It is probable, therefore, that BlrA, and its as yet undefined TCS partner, BlrB, form an activator/repressor of ß-lactamase transcription in Aeromonas spp.
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Acknowledgments |
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Notes |
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References |
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Received 21 March 2000; returned 5 June 2000; revised 20 June 2000; accepted 24 July 2000