Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
Received 18 November 2002; returned 15 January 2003; revised 3 March 2003; accepted 12 March 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Aeromonas, ß-lactamase, induction, two-component system, Blr, gene regulation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The paradigm for regulated ß-lactamase production in Gram-negative bacteria is the AmpC/AmpR system of Citrobacter freundii and Enterobacter cloacae.5 In these species, expression of a single ß-lactamase gene, ampC, is controlled negatively and positively by a LysR-type transcription factor, AmpR. Typical of such factors, the activity of AmpR is determined by a diffusible ligand, in this case, 1,6-anhydromuramyl pentapeptide (AHM-PP). AHM-PP is a product of peptidoglycan turnover and the substrate for the peptidoglycan recycling pathway found in Gram-negative bacteria.5 Normally, AHM-PP does not accumulate because it is broken down by an amidase, AmpD, destroying its ability to activate AmpR. Exposure to ß-lactams alters the balance in favour of intracellular accumulation of AHM-PP and ampC expression is induced.5
Coordinated expression of multiple ß-lactamases in Aeromonas spp. does not appear to involve an AmpR-like regulator, but rather, it may involve a two-component regulator (TCR) closely related to the CreBC TCR of Escherichia coli.4 CreBC is a global metabolic regulator that controls the expression of a number of genes in response to nutrient deprivation.6,7 The involvement of a TCR in Aeromonas spp. ß-lactamase induction was first demonstrated in A. jandaei AER14, where expression of a putative mutant form of a transcription factor, BlrA (related to the extended family of phosphorylation-dependent response regulators), was found to activate the expression of the three ß-lactamases.4 Involvement of a sensor kinase was not confirmed, but blrA was found to be immediately upstream from a gene, blrB, encoding what was predicted to be a sensor kinase. Unfortunately, blrB was truncated in the cloning process and only 73 bp, encoding the N terminus of BlrB, were recovered.
A. hydrophila strain T429125 produces three coordinately inducible ß-lactamases, AmpH, CepH and ImiH, with similar properties to those of A. jandaei and A. veronii bv. sobria.6 The genes encoding AmpH and CepH have been cloned and sequenced.6 In this study, the gene encoding A. hydrophila T429125 ImiH was cloned and sequenced, and the genetic contexts of the three ß-lactamase genes were investigated. The rationale employed was that genes encoding a bacterial transcription factor, and one of the genes whose expression it regulates, are often linked. Accordingly, the sequences upstream from ampH, cepH and imiH were examined for possible regulator genes. The aim was to learn more about the regulation of ß-lactamase expression in A. hydrophila and in Aeromonas spp. in general.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical and environmental isolates of Aeromonas spp. were collected from various countries and are described in Table 1, alongside the plasmids used in this study. T429125M1 and T429125M2 are ß-lactamase hyperproducing mutants of A. hydrophila T429125, and were isolated as described previously for an equivalent A. veronii bv. sobria 163a mutant, 163aM.2 Briefly, aliquots of an overnight broth culture were spread onto agar plates containing cefotaxime 2 mg/L (10 x MIC), and colonies on the plates following overnight growth were selected. All such mutants tested were found to hyperproduce ß-lactamases. All bacteria were grown on nutrient agar or in nutrient broth (Oxoid Ltd, Poole, UK). Meropenem was from Zeneca Pharmaceuticals (Macclesfield, UK) and other chemicals were from Sigma Chemical Co. or BDH (both of Poole, UK). Unless otherwise stated, enzymes for DNA manipulation were obtained from Invitrogen Life Technologies (Paisley, UK). PCR primers were from Sigma Genosys (Pampisford, UK).
|
All genetic manipulations were undertaken using standard methods as described previously.8 Purified A. hydrophila genomic DNA was digested to completion with EcoRI, and fragments were ligated into similarly linearized cloning vector, pSU18.9 The mixture of plasmids was transformed into E. coli DH5,10 and colonies containing a recombinant plasmid having the correct insert were selected by Southern colony blotting using a radiolabelled A. hydrophila AE036 cphA gene11 as a probe. One of the colonies selected contained a plasmid with a 3.5 kb insert, denoted pUB6067. Large quantities of pUB6067 were purified, 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.8 Sequence analysis was performed with the computer program Lasergene (DNA Star, Madison, WI, USA). The sequencing project was designed to reveal the sequence of imiH and its cloned 5'-proximal region only.
PCR to determine the full blrA sequence from A. hydrophila, and to confirm the relative positions of the blrABamp genes in Aeromonas spp.
Genomic PCR was performed using bacterial colonies as described previously.12 The primers used for amplification of A. hydrophila blrA were: blrA upstream forward (5'-GAAGGCATCGACGCTCAC-3'), derived from the previously published A. jandaei blrA sequence,4 and blrA reverse (5'-CTCTGTTCATGCCAGCTC-3'), derived from the A. hydrophila blrA 3' fragment obtained previously.6 Analysis of the relative positions of blrA, blrB and amp in various aeromonads was performed using the primers blrA upstream forward (as above), blrB forward (5'-CCATGCGTCGCCAGCTGGACG-3'), derived from the A. hydrophila blrB sequence, and amp reverse (5'-GCTCCTGTGGACTGATGG-3'), derived from the sequence of ampH.6 All PCR products were sequenced across the ends using the PCR primers to initiate sequencing as described previously,12 in order to confirm that the amplicons obtained corresponded to their target sequence. The blrA amplicon from A. hydrophila was sequenced more thoroughly so as to provide an accurate full-length sequence for this gene.
Cloning and sequencing of the ampH region from T429125M1 and T429125M2
The recovery of ampH from A. hydrophila T429125 on a 4.4 kb BamHI genomic fragment into plasmid pK1813 to produce construct pUB5972 has been previously described.6 The same procedure was used to clone ampH from T429125M1 and T429125M2, and so to produce constructs pUB5973 and pUB5975. Transformation of A. hydrophila T429125 with pUB5972, pUB5973 or pUB5975 was performed using the same conditions as for E. coli,8 with selection for resistance to kanamycin (30 mg/L). The procedures for sequencing the pUB5973 and pUB5975 inserts, and the methods of sequence analysis were as described above for imiH.
Preparation and assay of ß-lactamases
Bacterial strains were grown, cell extracts were produced and ß-lactamase assays were performed as described previously.6 One unit of ß-lactamase is defined as the amount of enzyme required to hydrolyse 1 nmol of substrate/min in the linear phase of the reaction at 25°C.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A. hydrophila T429125 produces three ß-lactamases: two serine active-site enzymes (AmpH and CepH) and a metallo-enzyme (ImiH), from genes that are unlinked.6 The sequences of ampH and cepH have been reported,6 but the sequence of imiH has not. This gene was cloned and imiH, together with the region immediately upstream of the gene, was sequenced as described in Materials and methods. The imiH sequence has been deposited with the EMBL database under accession number AJ548797. The predicted amino acid sequence of ImiH contains six mismatches compared with that of CphA from A. hydrophila AE03611 (Lys-86Gln; Glu-136
Arg, Val-165
Leu; Glu-186
Gln; Gln-187
Leu; Ile-228
Val), indicating that imiH is a T429125-specific cphA allele.
The genetic contexts of the three A. hydrophila T429125 ß-lactamase genes were investigated by examining the DNA sequences upstream of each gene for putative regulatory elements. Those of cepH6 and imiH gave little information of immediate interest; in particular, neither of the genes is linked to one encoding an AmpR-like transcription factor, nor to any other gene encoding a recognizable member of a transcription regulator family. In contrast, two genes of interest, designated blrA and blrB, were identified upstream from ampH (Figure 1).
|
|
|
Five additional Aeromonas spp. (A. veronii bv. sobria, A. jandaei, Aeromonas mediae, Aeromonas trota and Aeromonas salmonicida; Table 1) were tested for a blrABamp gene arrangement similar to that found in A. hydrophila T429125. DNA primers, based on sequences from A. jandaei AER14M were designed to target blrA (blrA upstream forward), and those based on sequences from A. hydrophila were used to target blrB (blrB forward) and amp (amp reverse) (Figure 1). Using the blrB forward and amp reverse primer set, PCR products of the expected 1.2 kb were obtained irrespective of the Aeromonas spp. from which the template genomic DNA was obtained, confirming the presence and relative positions of amp and blrB in each, of the species tested. Similarly, with the blrA upstream forward and amp reverse primer set, PCR products of 2.3 kb, as expected from the A. hydrophila sequence (Figure 1), were recovered using genomic DNA from all species. Sequencing of the ends of the PCR amplicons confirmed that they had amplified their correct targets in all of the aeromonads tested. Preliminary analysis of the sequence obtained suggests that there is no more than 15% divergence amongst the nucleotide sequences of the blrA and blrB genes from different aeromonads (data not shown).
Genetic and phenotypic comparisons between mutant and wild-type alleles of blrB
Substrate profiles were determined for the three cloned A. hydrophila ß-lactamase genes, cepH, ampH and imiH, when expressed from their native promoters in the E. coli strain DH5. The substrate profiles found in extracts of recombinant DH5
cultures are non-overlapping (Table 2), and these data allowed us to determine which ß-lactam to use for specific assays of each enzyme when produced in A. hydrophila T429125.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whilst we can say with some confidence that the Aeromonas BlrAB TCR regulates the coordinated expression of up to three unlinked ß-lactamase genes, the data shown in Table 3 indicate that the upregulation of enzyme production in A. hydrophila T429125M1 and T429125M2 compared with T429125 is not the same for each of the three ß-lactamases. The increase in ß-lactamase-specific activity seen for AmpH in the mutant is far less than for CepH, which is less than for ImiH. A similar phenomenon was seen in our previous study of A. hydrophila T429125, where, following ß-lactam challenge, the induction ratios of the three ß-lactamases fell into the same pattern; the expression of AmpH was increased much less than that of CepH, the induction of which was less than ImiH.6 Furthermore, A. hydrophila ß-lactamase expression is known to be regulated by the E. coli CreBC TCR when the ß-lactamase genes are cloned into E. coli,6,7 and the level of ß-lactamase induction following growth medium shift (which activates CreBC) follows the same pattern seen in Table 3; AmpH being activated far less than CepH, which is activated less than ImiH.7 It is thought that the regulation of AmpH, CepH and ImiH ß-lactamase gene expression in E. coli by CreBC is mediated by the cre-tag DNA sequence (TTCAC) found upstream of the promoter for each ß-lactamase gene. There is one copy of the cre-tag upstream of ampH, two upstream of cepH and three upstream of imiH.7 Given that the cre-tag may represent a CreB binding site, the total number of binding sites upstream of a ß-lactamase promoter might influence the level of control exerted by the regulator, explaining the differential growth medium-shift induction of the three genes. It is possible that BlrA (a close homologue of CreB)4 also uses the cre/blr-tag sequence to regulate the transcription of ampH, cepH and imiH, and that this is the reason for differential induction/overexpression of ß-lactamases seen in A. hydrophila T429125 (Table 3).
Increased expression of ß-lactamases in A. jandaei AER14M is thought to be through a mutation in blrA.4 However, the wild-type AER14 blrA sequence has not been reported, so there is no indication as to what the mutation might be. Indeed, overexpression of ß-lactamases in A. jandaei AER14 carrying blrA cloned from A. jandaei AER14M (the original reason why AER14M was thought to carry a blrA mutation) may well be due to the presence of blrA on a multi-copy plasmid, resulting in its overexpression. This phenomenon, of an overexpressed response regulator mimicking activation of the entire TCR, has recently been reported for E. coli PhoB, a response regulator very similar to BlrA.18 Accordingly, the actual mutation leading to ß-lactamase overexpression in A. jandaei AER14M is not certain. There might be an activating mutation in BlrB, as with A. hydrophila T429125M1, or there might be a mutation at an unlinked locus, as with A. hydrophila T429125M2. In this latter case, the result of the mutation is most likely to be either the overexpression of BlrAB, or overproduction/decreased removal of the BlrAB activating ligand. It is also possible, however, that the mutation in T429125M2 is in a separate regulatory system, that also regulates ß-lactamase expression. It is unlikely that BlrB interacts directly with ß-lactams to become activated, because it carries no recognizable ß-lactam binding motif, nor a motif associated with binding of any other ligand. Accordingly, more work is required to understand the mechanism of BlrB activation in Aeromonas spp. and the type of mutation found in T429125M2.
BlrAB represents the third distinct induction system for ß-lactamases, the others being the C. freundii AmpC/AmpR paradigm found in a number of Gram-negative bacteria,5 and the BlaI repressor-based, phosphorylation-independent TCR of Staphylococcus aureus and Bacillus spp.19 In these systems (with the possible exception of S. aureus), it is doubtful whether the primary role of the inducible ß-lactamases is cell protection from ß-lactam antibiotics. What the purpose of these enzymes is, however, is still speculative, although there is some evidence that they may play a role in determining cell morphology.2022 The fact that at least three different mechanisms have evolved to control the production of ß-lactamases argues, however, that these mechanisms must be important to the cells survival. Furthermore, these mechanisms probably evolved long before the clinical use of ß-lactam antibiotics.
It is not certain why a TCR has been employed by Aeromonas spp. to regulate ß-lactamase gene expression. It is possible, however, that a TCR is the most efficient way to coordinately regulate the expression of a number of unlinked genes. This seems to be the case with other TCRs, since most of them are known to regulate multiple unlinked genes,17 something that is not true of LysR-type regulators23 such as the paradigm ß-lactamase regulator, AmpR.5 Indeed, the coordinate regulation of multiple ß-lactamase genes has not been reported in bacteria other than Aeromonas spp. Other bacteria that have multiple ß-lactamases, such as Stenotrophomonas maltophilia, appear to use multiple induction mechanisms.24 Accordingly, given this property of coordinate control, the use of a TCR by Aeromonas spp. makes sense. It remains to be seen whether other non-ß-lactamase genes are also regulated by the BlrAB TCR.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
Present address. Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanuloke 65000, Thailand.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Walsh, T. R., Payne, D. J., MacGowan, A. P. & Bennett, P. M. (1995). A clinical isolate of Aeromonas sobria with three chromosomally mediated inducible ß-lactamases: a cephalosporinase, a penicillinase and a third enzyme, displaying carbapenemase activity. Journal of Antimicrobial Chemotherapy 35, 2719.[Abstract]
3 . Rasmussen, B. A., Keeney, D. Yang, Y. & Bush, K. (1994). Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER14M in Escherichia coli. Requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme. Antimicrobial Agents and Chemotherapy 38, 207885.[Abstract]
4 . Alksne, L. E. & Rasmussen, B. A. (1997). Expression of AsbA1, OXA-12 and AsbM1 ß-lactamases in Aeromonas jandaei AER14 is co-ordinated by a two-component regulon. Journal of Bacteriology 179, 200613.[Abstract]
5 . Weidemann, B., Dietz, B. & Pfeifle, D. (1998). Induction of ß-lactamases in Enterobacter cloacae. Clinical Infectious Diseases 27, S427.[ISI][Medline]
6
.
Avison, M. B., Niumsup, P., Walsh, T. R. & Bennett, P. M. (2000). The Aeromonas hydrophila AmpH and CepH ß-lactamases: derepressed expression in mutants of Escherichia coli lacking creB. Journal of Antimicrobial Chemotherapy 46, 695702.
7
.
Avison, M. B., Horton, R. E., Walsh, T. R. & Bennett, P. M. (2001). Escherichia coli CreBC is a global regulator of gene expression that responds to growth in minimal media. Journal of Biological Chemistry 276, 2695561.
8 . Sambrook, J., Fritsch., E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Vol. 1, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
9 . Martinez, E., Bartolome, B. & de la Cruz, F. (1988). pACYC184-derived cloning vectors containing the multiple cloning site and lacZ-alpha reporter gene of pUC8/9 and pUC18/19. Gene 68, 15962.[CrossRef][ISI][Medline]
10 . Hanahan, D. (1983). Studies on the transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166, 55780.[ISI][Medline]
11 . Massida, O., Rossolini, G. M. & Satta, G. (1991). The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-ß-lactamases. Journal of Bacteriology 173, 46117.[ISI][Medline]
12
.
Avison, M. B., von Heldreich, C. J., Higgins, C. S., Bennett, P. M. & Walsh, T. R. (2000). A TEM-2 ß-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia. Journal of Antimicrobial Chemotherapy 46, 87984.
13 . Pridmore, R. (1987). New and versatile cloning vectors with kanamycin resistance markers. Gene 56, 30912.[CrossRef][ISI][Medline]
14 . Amemura, M., Makino, K., Shinagawa, H. & Nakata, A. (1986). Nucleotide sequence of the phoM region of Escherichia coli: Four open reading frames may constitute an operon. Journal of Bacteriology 168, 294302.[ISI][Medline]
15
.
Record, M. T., Reznikoff, W. S., Craig, M. L., McQuande, K. L. & Schlax, P. J. (1996). Escherichia coli RNA polymerase (E70), promoters and the kinetics of the steps of transcription initiation. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 1, 2nd edn (Neidhardt, F. C., Curtiss, R., III, Ingram, J. L., Lin, E. C. C., Low, K. B., Magasanik, B. et al., Eds), pp. 792821. ASM Press, Washington, DC, USA.
16 . Yagar, T. D. & von Hippel, P. H. (1996). Transcriptional elongation and termination in Escherichia coli. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 1, 2nd edn (Neidhardt, F. C., Curtiss, R., III, Ingram, J. L., Lin, E. C. C., Low, K. B., Magasanik, B. et al., Eds), pp. 124175. ASM Press, Washington, DC, USA.
17 . West, A. H. & Stock, A. M. (2001). Histidine kinases and response regulator proteins in two-component signalling systems. Trends in Biochemical Sciences 26, 26976.
18
.
Ellison, D. W. & McCleary, W. R. (2000). The unphosphorylated receiver domain of PhoB silences the activity of its output domain. Journal of Bacteriology 182, 65927.
19 . Bennett, P. M. & Chopra, I. (1993). Molecular basis of ß-lactamase induction in bacteria. Antimicrobial Agents and Chemotherapy 37, 2732.
20 . Henderson, T. A., Young, K. D., Denome, S. A. & Elf, P. K. (1997). AmpC and AmpH, proteins related to the class C ß-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli. Journal of Bacteriology 179, 612231.[Abstract]
21 . Hochstadt Ozer, J., Lowery, D. L. & Saz, A. K. (1970). De-repression of ß-lactamase (penicillinase) in Bacillus cereus by peptidoglycans. Journal of Bacteriology 102, 5263.[Medline]
22 . Hochstadt Ozer, J. & Saz, A. K. (1970). Possible involvement of ß-lactamase in sporulation in Bacillus cereus. Journal of Bacteriology 102, 6471.[ISI][Medline]
23 . Schell, M. A (1993). Molecular biology of the LysR family of transcriptional regulators. Annual Reviews of Microbiology 47, 597626.[CrossRef]
24
.
Avison, M. B., Higgins, C. S., Ford, P. J., von Heldreich, C. J., Walsh, T. R. & Bennett, P. M. (2002). Differential regulation of L1 and L2 ß-lactamase expression in Stenotrophomonas maltophilia. Journal of Antimicrobial Chemotherapy, 49, 3879.