Genetic linkage of the penicillinase gene, amp, and blrAB, encoding the regulator of ß-lactamase expression in Aeromonas spp.

Pannika Niumsup*, Alan M. Simm, Kurshid Nurmahomed, Timothy R. Walsh, Peter M. Bennett and Matthew B. Avison§

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aeromonas hydrophila T429125, a human clinical isolate, possesses three coordinately inducible ß-lactamases encoded by ampH (class D ß-lactamase), cepH (class C ß-lactamase) and imiH (class B ß-lactamase). We report that upstream of ampH there are two genes, blrA and blrB, encoding a putative two-component regulatory system. PCR studies revealed the same blrAB–amp gene arrangement in all Aeromonas spp. isolates tested; namely, Aeromonas veronii bv. sobria, Aeromonas jandaei, Aeromonas mediae, Aeromonas salmonicida and Aeromonas trota. A dominant mutation in the predicted BlrB kinase domain results in ß-lactamase overexpression in A. hydrophila T429125, but in other ß-lactamase-overexpressing mutants blrAB remains intact. Relative to the parent strain, A. hydrophila T429125, ß-lactamase- overexpressing mutants show a clear hierarchy of increased ß-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following ß-lactam challenge of A. hydrophila T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each ß-lactamase gene: ampH (one), cepH (two) and imiH (three).

Keywords: Aeromonas, ß-lactamase, induction, two-component system, Blr, gene regulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aeromonas spp. typically produce two or three unrelated, inducible ß-lactamases;1 for example, Aeromonas veronii bv. sobria 163a produces three: a class 2d penicillinase, AmpS; an AmpC-like cephalosporinase, CepS; and a class 3 metallo-enzyme with carbapenemase activity, ImiS.2 Aeromonas jandaei AER14 also produces three enzymes: AsbA1, AsbB1 and AsbM1, falling into the same molecular classes as those of A. veronii bv. sobria.3 Production of the A. jandaei and A. veronii bv. sobria ß-lactamases is coordinated, in that expression of all three enzymes is induced by ß-lactams, and single-site mutations (according to the frequency that they are obtained) create derivatives that constitutively hyper-produce all three enzymes.2,4

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, media and reagents

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).


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Table 1.  Bacterial strains and plasmids used in this study
 
Cloning and sequencing of imiH, and the region immediately upstream of imiH from A. hydrophila T429125

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{alpha},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 blrAB–amp 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence analyses and gene contexts of the ß-lactamase genes of A. hydrophila T429125

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-86->Gln; 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).



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Figure 1. The blr gene cluster from A. hydrophila T429125. The sequence upstream of ampH on the insert of pUB5972 is depicted, together with the additional blrA sequence obtained by PCR. The widths of all genes are represented to scale. Intergenic regions are expanded to allow detailed analysis of each sequence. Putative ribosome binding sites are in bold type, putative promoter sequences are underlined and putative termination signals are italicized. Putative transcriptional units, and their direction of transcription, are marked with bold arrows. The positions where various primers used in this study bind are marked with numbered arrows. Primers are: 1, ‘blrA upstream forward’; 2, ‘blrA reverse’; 3, ‘blrB forward’; 4, ‘amp reverse’ (see text for details).

 
The blrA gene was truncated by the strategy used to clone ampH6 and only the terminal 60 bp of the gene were recovered. Subsequently, the remainder of blrA was recovered from A. hydrophila T429125 by PCR, using a negative primer anchored in the known 60 bp T429125 blrA sequence, and a positive primer derived from the A. jandaei blrA sequence4 (see Materials and methods) (Figure 1). Following sequencing of the amplicon obtained, BlrA from A. hydrophila T429125 was found to have 90% sequence identity with A. jandaei BlrA, indicating that the two genes are species-specific equivalents. The differences between the two Aeromonas spp. BlrA proteins are shown in Figure 2. The blrA sequence from A. hydrophila T429125 has been deposited with the EMBL database under accession number AJ548796.



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Figure 2. Amino acid sequence alignment of BlrA from A. hydrophila (A. h) and from A. jandaei (A. j). Mismatches are indicated by shading and gaps (-) are inserted as appropriate. The BlrA sequence from A. jandaei (A. j BlrA) comes from strain AER14M (Alksne & Rasmussen4); that from A. hydrophila (A. h BlrA) comes from strain T429125 (this report).

 
Thirty-four nucleotides downstream from A. hydrophila T429125 blrA is blrB, a 1422 nucleotide open reading frame encoding a putative peptide of 473 amino acids (predicted size 51.9 kDa) (Figure 1). BlrB is 65% identical to CreC (Figure 3), the sensor kinase of the E. coli CreBC TCR.14 The hydropathy profiles of CreC and BlrB are virtually identical (not shown), indicating that they are likely to have similar topologies; both are predicted to have two membrane-spanning regions, which for BlrB delineate a 155 amino acid putative periplasmic loop (Figure 3). Each has a long C-terminal cytoplasmic tail accommodating a putative histidine kinase domain (Figure 3). The 34 nucleotide blrA/blrB intergenic region contains no sequences typical of an E. coli {sigma}70-type promoter,15 nor an obvious transcription termination signal for blrA (Figure 1).16 It was hypothesized that the two genes are operonic, as is commonly found for genes encoding TCRs.17 A tetranucleotide inverted repeat followed by a poly(T) sequence is present 16 nucleotides downstream from the translational stop codon for blrB, and may function as a Rho-independent transcription terminator for the blrAB operon (Figure 1).16 Seventy-five nucleotides downstream of blrB, and similarly oriented, is ampH (Figure 1),6 which is known to have its own {sigma}70-type promoter that is active in E. coli.7



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Figure 3. Amino acid sequence alignment and properties of BlrB from A. hydrophila and CreC from E. coli. The individual regions making up the putative kinase domain (H-, N-, D/F- and G-boxes) are underlined, and aligned with the known consensus sequence for each domain (derived from West & Stock17). The putative autophosphorylated histidine (His265) is marked with an asterisk, and the residue mutated in T429125M1 is marked with a plus sign. The two predicted transmembrane helices are double underlined. Mismatches are indicated with shading.

 
Conservation of the blrAB–amp gene cluster in aeromonads

Five additional Aeromonas spp. (A. veronii bv. sobria, A. jandaei, Aeromonas mediae, Aeromonas trota and Aeromonas salmonicida; Table 1) were tested for a blrAB–amp 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{alpha}. The substrate profiles found in extracts of recombinant DH5{alpha} 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.


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Table 2.  ß-Lactamase substrate profile in extracts of E. coli DH5{alpha} recombinants expressing A. hydrophila ß-lactamasesa
 
A. hydrophila T429125 mutants T429125M1 and T429125M2, which hyperexpress their three ß-lactamases, were selected, and the coordinate overexpression of all three ß-lactamases was confirmed by assay using substrates specific to each enzyme (Table 3). To determine whether the mutation(s) responsible for ß-lactamase overexpression in T429125M1 and T429125M2 is within the blrB, chromosomal fragments carrying blrA (truncated), blrB and ampH were cloned from T429125M1 and T429125M2 genomic DNA into plasmid vector pK18 to give plasmids pUB5973 and pUB5975, and both inserts were sequenced. This revealed a single mutation in the T429125M1 sequence compared with the wild-type parent strain, T429125, an A to G transition, generating a Glu-318->Gly substitution in BlrB (Figure 3). We denote this blrB mutant allele, blrB2. No mutations were found in pUB5975, indicating that the blrB allele is intact in T429125M2. To determine whether there is a mutation(s) in blrA in T429125M1 or T429125M2, PCR sequencing of blrA from both mutants (using the ‘blrA upstream forward’ and ‘blrA reverse’ primer set) was performed. This showed that neither mutant carried an altered blrA allele. To confirm a role for the observed blrB mutation in A. hydrophila T429125M1, pUB5973 (blrB2) was introduced into A. hydrophila T429125. This caused T429125 to overexpress its three ß-lactamases (Table 3). In contrast, when equivalent wild-type DNA fragments from T429125 or T429125M2, on plasmids pUB5972 or pUB5975, were introduced into A. hydrophila T429125, expression of the ß-lactamases was not markedly altered (Table 3). So the blrB2 allele represents a dominant mutation that causes constitutive overproduction of ß-lactamases in A. hydrophila, and is highly likely to be the reason for ß-lactamase overexpression in A. hydrophila T429125M1. The mutation responsible for the ß-lactamase-overexpressing phenotype in T429125M2 does not reside in blrA or blrB, however.


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Table 3.  ß-Lactamase expression in A. hydrophila constructsa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The work reported here demonstrates the involvement of a histidine sensor kinase, BlrB, in the control of ß-lactamase production in A. hydrophila. Alksne & Rasmussen4 reported that coordinated expression of the three intrinsic ß-lactamases of A. jandaei might be controlled by a phospho-relay TCR, BlrAB, closely related to the CreBC TCR of E. coli, rather than an AmpR-like factor as is more generally the case in Gram-negative bacteria. Involvement of the response regulator, BlrA, was demonstrated, and linkage of blrA to a putative sensor kinase gene, blrB, was reported. In the present study, it has been shown that the A. hydrophila equivalent of blrA is part of a two-gene operon. The second gene of the operon, blrB, encodes a protein that is clearly involved in expression of the three ß-lactamases of A. hydrophila. The same blrAB locus is found in other species of Aeromonas, including A. jandaei, similarly linked to the gene encoding the OXA-type penicillinase, amp.

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 cell’s 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
 
We would like to thank Drs Gianni Rossolini (Siena), Sylvia Kirov (Hobart) and Adrian Elley (Sheffield) for their assistance in supplying Aeromonas strains, and Dr Jenny Jury, Rhiannon Murry and Jennie Douthwaite (Department of Biochemistry, University of Bristol) for DNA sequencing. ß-Lactamase induction research at the Bristol Centre for Antimicrobial Research and Evaluation is funded by the British Society for Antimicrobial Chemotherapy and the Wellcome Trust.


    Footnotes
 
* Correspondence address. Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. Tel: +44-117-928-7439; Fax: +44-117-928-8274; E-mail: Matthewb.Avison{at}bris.ac.uk Back

§ Present address. Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanuloke 65000, Thailand. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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