Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
Received 5 August 2003; returned 14 October 2003; revised 7 November 2003; accepted 17 November 2003
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Abstract |
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Methods: Genes linked to blrAB-ampH were cloned using standard methods; gene expression was measured by RTPCR or ß-lactamase assays; transcription start sites were determined by reversed-transcript analysis; cepH promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in Escherichia coli using the pBAD plasmid.
Results: The blrD gene, encoding a putative inner membrane protein, was found to be located downstream of blrAB-ampH. RTPCR analysis showed that blrD is part of the A. hydrophila blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of blrD) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp cepH cre/blr-tag dimer blocked BlrA-induced overproduction of cepH in E. coli.
Conclusions: This is the first report of non-ß-lactamase genes being co-ordinately regulated with a normally co-resident ß-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.
Keywords: blr regulon, cre regulon, CreBC, BlrD, cre/blr-tag
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Introduction |
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BlrAB is highly homologous to the Escherichia coli TCS, CreBC, previously known as PhoM orf2 and PhoM orf3,3,6,7 which has recently been shown to regulate the expression of cre-regulon genes.8 Of the conditions that have been tested, the expression of a cre-regulon gene is at its greatest (or least, depending on the gene) during growth of E. coli in minimal medium containing pyruvate as the carbon and energy source. Given the widespread functions encoded for by cre-regulon genes, it is likely that CreBC is a global regulator of intermediary metabolism.8 One of the members of the cre regulon is creD, a gene found immediately downstream of creBC.7,8 The expression of CreD is tightly regulated by CreBC.8 Interestingly, the three A. hydrophila ß-lactamase genes can also form part of the cre regulon (i.e. their expression comes under the control of CreBC) when they are cloned into the Cre+ E. coli strain, DH5.2,8
All known E. coli cre-regulon genes carry at least one direct repeat of a promoter-proximal DNA sequence motif, TTCAC, the cre/blr-tag, which may mediate CreBC-dependent transcriptional control. Identical sequences are found close to the promoters (as mapped in E. coli) of the three A. hydrophila ß-lactamase genes; a possible reason why they can be regulated by CreBC.8
It is possible that the cre/blr-tag DNA sequence is involved in BlrAB-dependent regulation of ß-lactamase expression in A. hydrophila, given the marked similarity between the proposed DNA binding domains of the transcription factors BlrA and CreB.3 Indeed, there is some evidence for the importance of the cre/blr-tag in regulating Aeromonas spp. ß-lactamase expression, but this is limited to observations that the fold-induction in response to ß-lactam challenge, or the fold-overexpression in a ß-lactamase overexpressing (BlrB2) mutant, of each ß-lactamase gene in A. hydrophila is proportional to the number of cre/blr-tag sequences found upstream of its promoter (as located in E. coli). The level of AmpH (one cre/blr-tag) induction/overexpression is less than that of CepH (two cre/blr-tags), which is less than that of ImiH (three cre/blr-tags). More significantly, however, this same correlation between number of cre/blr-tags and ß-lactamase induction/overexpression is found when looking at CreBC-dependent expression of A. hydrophila ß-lactamases in E. coli, where there is more direct evidence that cre/blr-tags are involved.2,6,8
In the project reported here, we set out to determine whether it is possible to collect experimental evidence that the cre/blr-tag sequence is not only genetically linked to BlrAB-regulated genes, but that it is important for the regulation of their expression by the transcription factor BlrA in A. hydrophila.
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Materials and methods |
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T429125M1 is a ß-lactamase hyperproducing mutant of A. hydrophila T4291252 carrying the blrB2 allele.6 The creABCD deletion E. coli strain RB2181 (supE44, lacY, tonA, thi, leu, str, phoR68, serB-thr)2,9 was used as a background for BlrA overexpression, and for checking the importance of the cre/blr-tag, because it lacks creBC, reducing the possible complications that come with using a cre+ strain.8 All bacteria were grown on nutrient agar or in nutrient broth (Oxoid Ltd, Basingstoke, UK). All chemicals were from Sigma Chemical Co. or BDH, both of Poole, Dorset, UK. Unless otherwise stated, enzymes for DNA manipulation were obtained from GIBCOBRL (Life Technologies, Paisley, UK). PCR primers were from Sigma Genosys (Pampisford, Cambridge, UK).
Isolation of total RNA, transcriptional start site determinations and RTPCR analysis
Total RNA isolation, RTPCR analyses and transcription start site determinations were carried out exactly as described previously.8 Transcriptional start point analyses used RNA isolated from A. hydrophila T429125M1. The primers used for reversed transcript analysis of transcriptional start points were: ampH (5'-GAGAAAGCAGCCGGTGGC-3'); cepH (5'-CATCCACCACAGCGTTCA-3'); imiH (5'-CTCGCCATCAGCACCACG-3'); blrD (5'-CTGGCAGCAGGTAGCGGTAGAGCTGCAG-3').
The A. hydrophila ribosomal protein S20 gene10 was chosen as the control for normalization of mRNA loading in each RTPCR as it was thought highly unlikely that its expression would be altered by the status of BlrAB. Following RTPCRs, amplicons were separated by gel electrophoresis and images of the ethidium bromide-stained gels were acquired using a Kodak Image Station 440CF (Kodak Ltd, Hemel Hempstead, UK) and analysed using the ImageQuant suite of programs (Molecular Dynamics Inc., CA, USA) to determine band intensities. In all cases, the intensities were normalized for loading using the intensity of the S20 band from each RNA preparation. The RTPCR primer sequences were: ampH (5'-GCGAACTCTATGTGCTCA-3' and 5'-GACCGTCGTGCTTGCCAG-3'); cepH (5'-CCACTGCTGGCCCTCGGC-3' and 5'-GGCACATCGAGATAAGTG-3');2 imiH (5'-GGTGCTGGAGGTGATCAA-3' and 5'-CCGTGCAGTGGCGAGTC-3'); blrA (5'-ATGCAAAAGAGAGTTATCTG-3' and 5'-TCATGCCAGCTCCAGGCTGT-3'); blrB (5'-CTGATCGACGGCCACCTC-3' and 5'-GCGAATGGCGGCCAGCGG-3');6 blrD (5'-CGCACAGTCACGGTGGAC-3' and 5'-GCCGATGAACAGCAGCGC-3') (this report); S20 (5'-CGCTCTTCAGTCAGAGAA-3' and 5'-GACAGGCGGCTCTTGTGA-3').10
Transient overexpression of BlrA in E. coli using pBAD
The A. hydrophila blrA gene was amplified by PCR according to a previously described method,11 using primers that span the entire coding sequence (i.e. 5'-ATGCAAAAGAGAGTTATCTG-3' and 5'-TCATGCCAGCTCCAGGCTGT-3'). The amplicon was TA-cloned downstream of the araBAD promoter into the overexpression plasmid, pBAD-TOPO (Invitrogen, Leek, Holland). E. coli TOP-10 cells (Invitrogen) carrying pBAD recombinants containing correctly oriented inserts were selected according to the manufacturers instructions. One pBAD recombinant plasmid carrying blrA was named pUB6072. This plasmid was purified and used to transform E. coli RB2181 cells (creABCD)2,9 to ampicillin resistance (50 mg/L). Transient overexpression of BlrA was induced by adding 0.2% w/v arabinose to nutrient broth growing cultures at a starting OD600 of 0.3 AU. Induction was continued for 2 h before cell extraction and assay of the appropriate reporter.
Development of cepH reporter constructs and assay of ß-lactamase
Two PCR amplicons were produced,11 having 5' termini anchored at the immediate 5' or 3' end of the cepH cre/blr-tag dimer and 3' termini downstream of the cepH coding region.2 The two forward primers used were: cre/blr-tag forward (5'-GGCTGGTGACTTCACACA-3') and cre/blr-tag forward (5'-CCAGGCTCATGCCCG-3') together with a single negative primer: cepH reverse (5'-GCTACCAACCCTCTATGC-3'). The two PCR amplicons were TA-cloned into pCR4.1 (Invitrogen) according to the manufacturers instructions, and the inserts sequenced. Correct inserts were removed by EcoRI digestion and subcloned into the pMB1 (i.e. pBAD oriC) compatible cloning vector, pSU18.12 The resultant correctly oriented recombinant plasmids were named pUB6070 (cre/blr-tag) and pUB6071 (
cre/blr-tag) and were used to transform E. coli RB2181 to chloramphenicol resistance (30 mg/L). Extraction and assay of ß-lactamase activity was carried out as described previously2,13 using ceftazidime as a specific CepH substrate because this compound is not hydrolysed by the TEM ß-lactamase carried by AmpR TA-cloning vectors such as pBAD.11,14
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Results |
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The E. coli cre-regulon gene creD, encoding an inner membrane protein of unknown function, is encoded downstream from creBC.7,9 We therefore sequenced downstream of blrAB-ampH on the pUB5972 insert2 to determine whether a creD homologue is present (EMBL accession number AJ276632). One hundred and eighteen nucleotides downstream from ampH is blrD, encoding a predicted 449 amino acid protein of 49.5 kDa (Figure 1). The deduced amino acid sequence of BlrD shows 35% identity to E. coli CreD.7,9 BlrD, like CreD, is hydrophobic, with hydropathy profiles15 of the two proteins being very similar (Figure 2). Hence, BlrD is predicted to possess six transmembrane domains (TMDs): one in the N-terminal and five in the C-terminal portions of the protein. TMDs 1 and 2 of both CreD and BlrD flank what are predicted to be large periplasmic loops (169 aa in CreD and 179 aa in BlrD) (Figure 2). The final transcriptional terminator in the insert of pUB5972 is 50 bp downstream of blrD, and no genes of immediate interest are found in the remaining 457 bp of the insert. This suggests that blrD is the 3' proximal gene in the blrA, blrB, ampH, blrD gene cluster (Figure 1).
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Previous transcript start site analysis in E. coli has implicated the cre/blr-tag sequence, TTCAC in CreBC-dependent regulation of cloned A. hydrophila ß-lactamase gene expression.8 Transcriptional starts for the three ß-lactamase genes were determined in A. hydrophila T429125M1, and are very similar to those found in E. coli (Figure 3). We cannot provide an explanation for the small differences seen, though they probably reflect differences in promoter specificity of the A. hydrophila and E. coli RNA polymerase complexes.17 It was thought likely, however, that the cre/blr-tags are sufficiently closely associated with the native A. hydrophila ß-lactamase gene promoters, that they could be responsible for BlrAB-dependent regulation of ß-lactamase expression in A. hydrophila. Accordingly, we predicted that the independent activation of transcription from the blrD promoter would be due, similarly, to the presence of at least one upstream cre/blr-tag. Following transcriptional start site analysis for blrD using mRNA from A. hydrophila T429125M1, three promoter-proximal cre/blr-tags were located (Figure 3).
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It has been previously shown that overexpression of BlrA stimulates ß-lactamase expression in A. jandaei3 so we tested whether BlrA overexpression in E. coli could induce gene expression in a manner dependent upon the cre/blr-tag. To this end, blrA was amplified by PCR and TA-cloned into the pBAD-TOPO vector which supports arabinose inducible transcription of the cloned insert; the plasmid was designated pUB6072. The reporter constructs used to measure cre/blr-tag gene expression used the cepH ß-lactamase gene and its native promoter. The gene was amplified by PCR, with (pUB6070) or without (pUB6071) its 16 bp cre/blr-tag dimer and both amplicons were TA-cloned into the cloning vector, pCR4.1, and then subcloned into the cloning vector, pSU18.12 E. coli RB2181 (a creABCD cluster deletion mutant)9 was transformed with one of the reporter constructs and pUB6072 (expressing BlrA) or empty pBAD vector as a control. Assays of CepH activity were carried out using nutrient broth growing cultures in the presence or absence of 0.2% (w/v) arabinose for 2 h to stimulate expression of BlrA from pUB6072. These results (Table 2) clearly indicate that the overexpression of BlrA stimulates the expression of CepH in E. coli, but only if the cre/blr-tag is present upstream of cepH.
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Discussion |
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All of the Aeromonas spp. ß-lactamase genes cloned to date have at least one copy of the cre/blr-tag sequence, TTCAC, which was originally found to be associated with genes regulated by the CreBC TCS in E. coli.8 Indeed, A. hydrophila ampH, cepH and imiH can be regulated by the CreBC TCS when cloned into the Cre+ E. coli strain, DH5.8 Transcriptional start point analyses put cre/blr-tag sequences in the correct vicinity, with respect to the ß-lactamase gene promoters in A. hydrophila, that would be expected of true regulatory sequences; a property shared by the blr-regulon gene, blrD. General evidence that this sequence is involved in regulated ß-lactamase expression comes from the observation that the greater the number of cre/blr-tags found upstream of a blr-regulon gene, the more highly expressed that gene is during ß-lactam challenge, or in a ß-lactamase overexpressing mutant. The most significant direct piece of evidence for the importance of the cre/blr-tag sequence presented here, comes from the cre/blr-tag deletion analysis. This experiment was carried out in a surrogate cre negative E. coli host so as to make interpretation of the results less complex, and clearly showed that BlrA cannot stimulate cepH expression in the absence of the 16 bp cre/blr-tag dimer, but does so if the cre/blr-tag is present upstream of cepH. These data provide considerable evidence for a role of this sequence in BlrAB-dependent regulation of blr-regulon gene expression in Aeromonas spp. The sequence is sufficient (the sequence upstream of cepH in pUB6070 contains no sequences other than the cre/blr-tag dimer and the promoter) and necessary (in pUB6071, only the cre/blr-tag is deleted, leaving the promoter intact) for BlrA-dependent activation of gene expression. The data also provide confirmatory evidence that BlrA is capable of regulating Aeromonas spp. ß-lactamase genes in the absence of BlrB. This is important, because interpretation of our previous studies using mutants of BlrB6 might have been complicated if BlrB can stimulate the phosphorylation of response-regulators other than BlrA. The data presented in Table 2 confirm that if such cross-talk does occur, it is not the only reason for ß-lactamase up-regulation in a BlrB mutant, and, as such, confirms a role for the BlrAB TCS acting in concert.
It is highly likely that BlrAB represent the Aeromonas spp. incarnation of E. coli CreBC. Differences in sequence between CreC and BlrB6 are likely to be the reason for the different stimulatory signals recognized by the two proteins. In contrast, the large degree of sequence similarity found, particularly in the proposed DNA binding domain of CreB and BlrA,3 may well explain how both can regulate the expression of genes having identical, upstream cre/blr-tag DNA sequences. Whilst CreBC regulates the expression of genes encoding metabolic functions, BlrAB regulates ß-lactamase gene expression. It is not clear why these two apparently disparate sensory mechanisms share identical regulatory mechanisms, particularly since the regulated genes are different in each case.
We have not set out to prove that BlrA binds directly to the cre/blr-tag during this study, but this is the most obvious explanation for the observations reported. This hypothesis is further supported by the finding that the cre/blr-tag dimer upstream of cepH (TTCACnnnnnnTTCAC) is very similar in architecture to the known binding site (TGTCAnnnnnnTGTCA) of the BlrA homologue, PhoB.3,18
Taken together, the data presented here expand our understanding of the complex ß-lactamase induction mechanism that seems to be unique to members of the genus Aeromonas. There are still many questions to answer, however; not least: what is the signal that communicates the presence of ß-lactams to the BlrAB TCS?
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Acknowledgements |
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Footnotes |
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Present address. Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanuloke 65000, Thailand.
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