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Regulation of class D -lactamase gene expression in Ralstonia pickettii

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le Kremlin-Bicêtre, France

Correspondence
Thierry Naas
thierry.naas{at}bct.ap-hop-paris.fr

	ABSTRACT

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Ralstonia pickettii, an environmental bacterium that may also be responsible for human infections, produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. In order to study the molecular basis of the induction process of these oxacillinase genes, the induction kinetics, the promoter/operator regions necessary for expression and induction, and the role of several ORFs located upstream and downstream of the bla_OXA genes were investigated. The -lactamase production reached a maximal level after 1 h induction, returned to its basal level within the following 3 h and was then again inducible. Using 5'RACE experiments, the promoter sequences of both oxacillinases were determined. These sequences showed weak promoter activities, which could, however, be increased approximately 200-fold by mutating the –35 promoter sequence. Deletion of the sequences located upstream of the promoter regions did not modify the basal -lactamase expression in R. pickettii, but resulted in the lack of induction. A minimum of 240 and 270 bp upstream of the transcription initiation sites was required for inducible expression of the bla_OXA-22 and bla_OXA-60 genes, respectively. Analysis of the genetic environment of both bla_OXA genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of bla_OXA-60 and divergently transcribed, abolished induction of both -lactamases. ORF-RP3, which encoded a polypeptide of 532 aa with an estimated molecular mass of 58.7 kDa, displayed no obvious sequence homology with known regulatory proteins. Trans-complementation of ORF-RP3 restored the basal and inducible expression of both oxacillinase genes, indicating that the induction of both enzymes was related to the presence of ORF-RP3. In addition to the loss of induction, inactivation of the ORF-RP3 in R. pickettii resulted in a complex pleiotropic phenotype, with increased lag phase and reduced survival after heat exposure, suggesting that ORF-RP3 might be a global regulator involved in unrelated regulatory pathways.

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the bla_OXA-22 genetic environment reported in this paper is AF064820.

	INTRODUCTION

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Ralstonia pickettii is a non-fermenting Gram-negative rod that is isolated from water, soil, plants, fruits and vegetables (Giligan, 1995). It is rarely involved in nosocomial septicaemia and tissue infections (Chen et al., 1995; Vershraegen et al., 1985). Most of the infections are traced to contamination of parenteral fluids or of medical equipment (Chetoui et al., 1997; Dimech et al., 1993; Kahan et al., 1983; Raveh et al., 1993).

All the tested R. pickettii strains possess two chromosomally located and inducible Ambler class D -lactamases (oxacillinases; Ambler et al., 1991): OXA-22 (Nordmann et al., 2000) and OXA-60 (Girlich et al., 2004b). Oxacillinases (oxacillin-hydrolysing -lactamases) usually hydrolyse oxacillin, methicillin and cloxacillin better than benzylpenicillin and their activity is inhibited by NaCl (Naas & Nordmann, 1999; Bush et al., 1995). While most of the oxacillinases are plasmid-mediated, several chromosomally encoded oxacillinases have been reported in environmental species (Héritier et al., 2004; Poirel et al., 2004; Salanoubat et al., 2002) and also in clinically relevant Gram-negative bacteria, such as Pseudomonas aeruginosa, Aeromonas sp. and Acinetobacter baumannii (Alksne & Rasmussen, 1997; Girlich et al., 2004a; Rasmussen et al., 1994; Héritier et al., 2005).

Although most of the -lactamase genes are not regulated, modulation of the expression of antibiotic-resistance genes can occur by insertion of insertion sequences (IS), which bring promoters located in or near their inverted-repeat (IR) sequences (Aubert et al., 2003). Inducible expression of the chromosomally encoded class C -lactamases (cephalosporinases) of Gram-negative bacteria is usually regulated by LysR-type elements (Lindberg et al., 1985; Lindberg & Normark, 1987; Lindquist et al., 1989). These proteins act as negative regulators in the absence of -lactam inducers and as positive regulators in the presence of -lactam inducers (Bennett & Chopra, 1993). LysR-type elements are also involved in inducible expression of carbapenem-hydrolysing class A -lactamases such as NmcA (Naas & Nordmann, 1994) and SmeA (Naas et al., 1995). As for ampC-ampR systems of Enterobacter cloacae (Lindberg & Normark, 1987), Citrobacter freundii (Bartowsky & Normark, 1991; Lindberg et al., 1985), Yersinia enterocolitica (Seoane et al., 1992), P. aeruginosa (Lodge et al., 1990), Morganella morganii and Hafnia avei (Poirel et al., 1999; Girlich et al., 2000), the regulatory genes are located immediately upstream of the structural -lactamase genes and divergently transcribed. By contrast, in the smeR-smeA system from Serratia marcescens (Naas et al., 1995), and nmcA-nmcR from Ent. cloacae (Naas & Nordmann, 1994), the LysR-like proteins act as positive regulators both in the absence and in the presence of a -lactam inducer. Such LysR-like proteins acting as positive regulators are also involved in the regulation of -lactamases in Gram-positive bacteria such as Streptomyces cacaoi (Urabe & Ogawara, 1992). BlaP and BlaZ -lactamase secretion is also induced in Bacillus licheniformis and Staphylococcus aureus, respectively, in the presence of exogenous inducers (Joris et al., 1994; Philippon et al., 1998). In both strains, the regulatory genes blaI, blaR1 and blaR2 are involved in the derepression of blaP or blaZ genes. These regulatory genes encode a cytoplasmic repressor (BlaI) that maintains a low level of -lactamase expression in the absence of antibiotic and a penicillin sensory transducer (BlaR1). The additional regulatory gene, blaR2, is not yet identified, but it is believed to play an essential role in the signal transfer process (Filee et al., 2002). -Lactamase synthesis regulatory genes blaI and blaR1 in B. licheniformis are similarly located upstream of the -lactamase gene and divergently transcribed (Kobayashi et al., 1987).

The only known regulation system of chromosomally encoded class D -lactamases is that of Aeromonas spp. (Alksne & Rasmussen, 1997; Iaconis & Sanders, 1990; Rasmussen et al., 1994). Coordinated expression of multiple -lactamases in Aeromonas spp. does not involve an AmpR-like regulator, but a two-component system (TCS) closely related to the CreBC TCS of Escherichia coli (Alksne & Rasmussen, 1997; Avison et al., 2004). -Lactamase induction in Aeromonas spp. depends upon the expression of the transcription regulator BlrA (related to the family of phosphorylation-dependent response regulators) and a sensor kinase BlrB. The genes blrA and blrB, encoding the corresponding proteins, are located immediately upstream of the oxacillinase-encoding ampH gene in Aeromonas hydrophila (Niumsup et al., 2003; Avison et al., 2004).

In the present study we investigated the molecular mechanism involved in the expression of the bla_OXA-22 and bla_OXA-60 -lactamase genes. We compared the induction kinetics with those of two well-known regulation systems (AmpC from C. freundii and Amp, Cep and Imi from Aeromonas spp.). We characterized the promoters of the bla_OXA-22 and bla_OXA-60 genes and determined the DNA sequence necessary for induction and expression. Furthermore, as for other systems, we investigated the genetic environment of both -lactamase genes in order to identify potential regulators. We characterized an open reading frame (ORF-RP3) that is involved in the expression and induction of both -lactamase genes.

	METHODS

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Bacterial strains and plasmids.
R. pickettii clinical isolate PIC-1 has been previously described (Girlich et al., 2004b; Nordmann et al., 2000). R. pickettii PIC-1OXA-22 and PIC-1OXA-60 isogenic mutants, lacking OXA-22 and OXA-60 respectively, were obtained as described previously (Girlich et al., 2004b). A. hydrophila CIP76.14 and C. freundii P478 strains were used in induction kinetics experiments (Pasteur Institute). E. coli DH10B was used as a host for cloning experiments. The kanamycin-resistant pPCRBluntII-TOPO plasmid (Invitrogen) was used as cloning vector and as a suicide vector for gene inactivation in R. pickettii PIC-1 strain since its ColE1 origin of replication restricts its host range to E. coli and a few other enterobacterial species. The tetracycline-resistant multicopy plasmid pLAFR3 was used as cloning vector in R. pickettii (Staskawicz et al., 1987). Bacterial cultures were grown in trypticase soy (TS) broth at 37 °C for 18 h unless indicated and were monitored by optical density at 600 nm using an Ultrospec 2000 (Amersham Biosciences). Plasmids were introduced in R. pickettii by electroporation (Gene pulser, Bio-Rad) with the same technique as that used for E. coli (Sambrook & Russell, 2001).

Induction studies and -lactamase assay.
Inducibility of the -lactamase content from each R. pickettii culture was tested in TS broth at 37 °C using the induction protocol with several concentrations of imipenem or cefoxitin as described (Poirel et al., 1999). These -lactam inducers are known to be good inducers of -lactamase expression (Poirel et al., 1999). For -lactamase induction kinetics, -lactam inducers were added to 200 ml TS broth cultures of R. pickettii PIC-1, A. hydrophila CIP76.14 and C. freundii P478 in exponential phase (OD₆₀₀ value of 0.7). Ten millilitres of the culture was collected by centrifugation during the growth and the -lactamase activity in crude extracts was determined as described by Poirel et al. (1999). The -lactamase activity was monitored over a period of 75 h. Eighteen hours after imipenem addition, cells were washed, diluted 1 : 100 in fresh medium, and the growth was continued for an additional 48 h. One unit of -lactamase activity was defined as the amount of enzyme that hydrolysed 1 µmol benzylpenicillin or nitrocefin per minute. The total protein content was measured using the DC Protein assay kit (Bio-Rad).

Plasmid extraction, cloning and PCR experiments.
Recombinant plasmid DNA was prepared by using Qiagen midi columns (Coger). All enzymes for DNA manipulations were used according to the recommendations of the supplier (Amersham Biosciences). Unless specified, standard molecular techniques were used (Sambrook & Russell, 2001). Whole-cell DNA of R. pickettii PIC-1 was extracted as previously described (Nordmann et al., 2000) and used as template for PCR amplification. For each PCR experiment, 500 ng total DNA was used in a standard PCR reaction mixture supplemented with 10 % (v/v) DMSO (Girlich et al., 2004b; Sambrook & Russell, 2001).

The recombinant plasmid pC2, containing the bla_OXA-60 gene and surrounding sequences, has been previously described (Girlich et al., 2004b). In order to obtain a recombinant plasmid with a flanking DNA sequence upstream of the bla_OXA-22 gene in E. coli DH10B, a ligation-mediated PCR (LMPCR) was developed (Prod'hom et al., 1998). Genomic DNA from R. pickettii PIC-1 was digested with SacII restriction endonuclease, blunt-ended with Pfu polymerase and ligated into pPCR-BluntII TOPO (Invitrogen), used as a linker for subsequent amplification with a primer pair recognizing the linker, M13-40 universal primer and OXA-22 intINV-1, complementary to the bla_OXA-22 gene sequence (Table 1).

A PCR product of 1684 bp, including the complete sequence of the rp3 gene, was generated using primers RP3A and RP3B (Table 1), located at each end of the rp3 gene of R. pickettii PIC-1 (Girlich et al., 2004b). PCR amplicon of the entire ORF-RP3 was then cloned into plasmid pPCRBluntII-TOPO, as recommended by the manufacturer (Invitrogen) and expressed in E. coli DH10B. The cloned insert was then removed by EcoRI (Amersham Biosciences) restriction and subcloned into the EcoRI-digested shuttle vector pLAFR-3 (Staskawicz et al., 1987), which replicates in both R. pickettii and E. coli. The recombinant plasmid, named pLAF-RP3, was introduced into R. pickettii PIC-1 by electroporation as previously described (Girlich et al., 2004b).

Gene inactivation.
Recombinant plasmids pORF-RP2, pORF-RP3, pORF-RP4, pORF-D and pORF-E were used as suicide vectors for homologous recombination in R. pickettii PIC-1 as previously described (Girlich et al., 2004b). Strains deficient in ORF-RP2, -RP3, -RP4, -D and -E and in the sequences upstream of the bla_OXA-22 and bla_OXA-60 genes obtained after a single recombination event were selected onto TSA plates containing kanamycin (Kan, 30 µg ml^–1). The disruption of the targeted genes in R. pickettii was verified by PCR.

Promoter sequence determination.
Different sizes of DNA regions containing the promoter sequence were constructed to dissect the regulatory region and to determine a DNA fragment that carries an active promoter and/or an active operator. R. pickettii PIC-1 total DNA was used as template in PCR experiments with primers located upstream of the bla_OXA-22 gene on the one hand (MR1, MR2, MR3, MR4, MR5) and one internal primer, OXA-22C, located in the bla_OXA-22 gene on the other hand (Table 1). The amplified fragments were blunt-ended with Pfu DNA polymerase and ligated into pPCR-BluntII TOPO (Invitrogen) in E. coli DH10B. The resulting recombinant plasmids, pMR1, pMR2, pMR3, pMR4 and pMR5, were used as suicide vectors for homologous recombination in R. pickettii PIC-1OXA-60. All constructs contained the putative ⁷⁰ bla_OXA-22 promoter intact or with an E. coli ⁷⁰ –35 promoter-consensus sequence TTGACA (pMR5) (Lisser & Margalit, 1993).

Similar constructs were made with DNA fragments located upstream of bla_OXA-60. R. pickettii PIC-1 total DNA was used as template in PCR experiments with primers located upstream of the bla_OXA-60 gene on the one hand (MR6, MR7, MR8, MR9, MR10) and one internal primer, OXA-60E, located in the bla_OXA-60 gene on the other hand (Table 1). All constructs contained the putative ⁷⁰ bla_OXA-60 promoter, intact or with a E. coli ⁷⁰ –35 promoter-consensus sequence TTGACA (pMR10). The constructs with bla_OXA-60 minimal regions (MRs) were introduced in R. pickettii PIC-1OXA-22, an isogenic mutant of R. pickettii PIC-1 lacking bla_OXA-22 gene expression. The level of -lactamase expression from the different isogenic mutants was determined as described by Philippon et al. (1997).

Mapping the transcription start sites.
Reverse transcription and rapid amplification of cDNA ends (RACE) were performed with the 5'RACE system version 2.0 (Invitrogen). Five micrograms of total RNAs extracted from an imipenem-induced culture of R. pickettii PIC-1 (Qiagen) and the OXA-22GSP1 and OXA-22GSP2 antisense bla_OXA-22 gene-specific primers were used to determine the transcription initiation site of the bla_OXA-22 gene (Table 1).

Site-directed mutagenesis.
Since the identified sequence upstream of the bla_OXA-22 gene contained a tandem repeat GTTAC-n₄-GTTAC similar to the ‘cre/blr-tag’ TTCAC-n₆-TTCAC from Aeromonas sp. (Fig. 2, Avison et al., 2001) at positions –62 to –49 relative to bla_OXA-22 (+1), a site-directed mutagenesis protocol was used as described by the manufacturer (Quick Change site-directed mutagenesis kit; Stratagene) for a deletion experiment. Recombinant plasmid pMR2, containing the MR upstream of the bla_OXA-22 gene necessary for the inducibility of the expression of the gene and a truncated copy of the bla_OXA-22 gene, was used as the template with primers Am22mut-1 and Am22mut-2 to generate recombinant plasmid pAm22mut (Table 1). Introduction of this suicide plasmid by electroporation into R. pickettii PIC-1OXA-60 resulted in deletion of the tandem repeat GTTAC-n₄-GTTAC upstream of the chromosomal copy of the bla_OXA-22 gene.

View larger version (4K): [in this window] [in a new window]   Fig. 2. Nucleotide sequence of the promoter of the blaOXA-22 and blaOXA-60 genes and upstream-located regions. The conserved regions (–35, –10 and +1) for RNA polymerase binding sites and ATG initiation codons of the -lactamase genes are in bold italic. The transcriptional start site was determined experimentally with a poly-dC tailing reaction (5'RACE technique), leading to an unspecified determination for the blaOXA-60 gene start site (the G or the first C are possible +1). Hyphenated tandem repeats GTTAC-n4-GTTAC are bold and underlined and nucleotide identities are marked with a star.

DNA sequencing, DNA and protein analyses.
PCR-generated fragments, purified using Quiaquick PCR purification spin columns (Qiagen), and the inserts of the recombinant plasmids were sequenced on both strands on an ABI 3100 automated sequencer (Applied Biosystems). The nucleotide and the deduced protein sequences were analysed with software available over the Internet at the National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Multiple nucleotide and protein sequence alignments were carried out online by using the program CLUSTALW available over the Internet at the University of Cambridge (http://www.ebi.ac.uk/clustalW/).

	RESULTS AND DISCUSSION

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Induction kinetics
Preliminary studies on induction had suggested that the -lactamase expression in R. pickettii was inducible and that both oxacillinases might be co-regulated (Table 2, Girlich et al., 2004b). Imipenem and cefoxitin do not behave in the same way in the induction of both oxacillinases, probably because imipenem only is hydrolysed by OXA-60. In fact, imipenem (1 µg ml^–1) is the best inducer of OXA-22 (no hydrolysis) and cefoxitin (5 µg ml^–1) is the best inducer of OXA-60 (no hydrolysis) (Table 2, Girlich et al., 2004b). In order to investigate the kinetics of induction and to rule out in vitro selection of hyper-producing strains, induction experiments were performed over 75 h. After induction, a rapid increase of the -lactamase activity was measured (maximal level reached after 1 h induction). Four hours after induction, the -lactamase expression dropped significantly to reach almost its basal level. After 18 h, the basal level was reached and -lactamase expression was still inducible upon reinduction (data not shown). These results showed that addition of a -lactam inducer resulted in reversible -lactamase expression in R. pickettii (Fig. 1). The OXA-60 and OXA-22 induction kinetics were similar to those observed with imipenem-induced (1 µg ml^–1) cultures of A. hydrophila CIP76.14 and cefoxitin-induced (10 µg ml^–1) cultures of C. freundii P478 (Fig. 1). This similarity in the induction behaviour suggested that these -lactamase genes might be regulated by a system that could be related to any of those described in the two other species, but is not the result of selection of a derepressed mutant (Fig. 1).

View this table: [in this window] [in a new window]   Table 2. -Lactamase activity of R. pickettii PIC-1 and isogenic mutants PIC-1OXA-22 and PIC-1OXA-60, deficient in OXA-22 and OXA-60, respectively.

View larger version (6K): [in this window] [in a new window]   Fig. 1. -Lactamase levels produced by cultures of R. pickettii PIC-1 (), A. hydrophila CIP76.14 () and C. freundii P478 () cultures after induction. Imipenem (1 µg ml–1) was used as the -lactam inducer for R. pickettii PIC-1 and A. hydrophila. Cefoxitin (10 µg ml–1) was used as the -lactam inducer for C. freundii P478 cultures. Benzylpenicillin (100 µM) was used as substrate.

Mapping of the transcription start site of bla_OXA-22
Using 5'RACE PCR experiments, the site of initiation of transcription of the bla_OXA-22 gene was mapped in R. pickettii PIC-1 and the deduced promoter region was compared with that of the bla_OXA-60 gene (Girlich et al., 2004b) (Fig. 2). The nucleotide sequence of the 5'RACE PCR product showed that transcription started at the A located 34 bp upstream of the bla_OXA-22 translation start codon. Upstream of this transcriptional start point (TSP, +1), a –35 promoter sequence CTGCAG, was found, separated by 17 bp from a –10 promoter sequence, TACGCT (Fig. 2). For the bla_OXA-60 gene, the transcription started at the cytosine located 55 bp upstream of the bla_OXA-60 translation start site (Girlich et al., 2004b). A putative ⁷⁰ promoter was identified at position 64–92 nucleotides (TGGCCG-n₁₇-TACGAT) upstream of the bla_OXA-60 translation start site (Fig. 2). Promoter sequence analysis revealed in both cases a –35 promoter sequence that diverged from the E. coli ⁷⁰ promoter-consensus sequence TTGaca (Lisser & Margalit, 1993).

Characterization of the minimum promoter/operator region
Homologous recombination was used to identify the regulatory region and to determine a DNA fragment that carries an active bla_OXA-22 and bla_OXA-60 promoter (Fig. 3a). Different sizes of DNA regions containing the promoter sequence were constructed. The constructs carried either the largest portion of the bla_OXA-22 regulatory region (pMR1) or 5' deletion derivatives, resulting in regions B (pMR2), C (pMR3) and D (pMR4) (Fig. 3b). All constructs contained the putative ⁷⁰ bla_OXA-22 promoter. Since the expression of the bla_OXA-22 and bla_OXA-60 genes was inducible, the constructs with bla_OXA-22 minimal regions (MRs) were introduced into R. pickettii PIC-1 OXA-60, an isogenic mutant of R. pickettii PIC-1 lacking a bla_OXA-60 gene. The level of -lactamase activity from the different isogenic strains indicated low-level and inducible expression of OXA-22 only when the promoter sequence followed a minimal sequence of 240 bp. Shortening the promoter upstream region by 110 bp (from region A to region B, Fig. 3) resulted in the loss of 65 % of the -lactamase activity after induction. Further shortening by 81 bp (from region B to region C or D, Fig. 3) resulted in loss of inducibility of bla_OXA-22 gene expression.

View larger version (20K): [in this window] [in a new window]   Fig. 3. -Lactamase expression in R. pickettii PIC-1 isogenic strains. Schematic representation of the insertion-inactivation of R. pickettii sequences. Homologous recombination was used to analyse the regulatory region and to determine a DNA fragment that carries active blaOXA-22 and blaOXA-60 promoter. The open arrow indicates the direction of transcription of the non-modified blaOXA gene. In R. pickettii PIC-1 isogenic mutants, harbouring inserted plasmids pMR1 to 10, which conferred resistance to kanamycin (Kanr), the sequence of pPCR-Blunt TOPO is grey and the cloned fragment is hatched. Upon insertion, recombination yields two copies of the blaOXA gene, one truncated and the other intact, downstream of a restricted promoter region (a). The cloned fragments consisted of different sizes of DNA regions containing the promoter sequence of the blaOXA-22 (b) or blaOXA-60 (c) gene and the corresponding truncated blaOXA gene. -Lactamase expression was determined with each construction in R. pickettii PIC-1OXA-60 (b) and in R. pickettii PIC-1OXA-22 (c) before and after imipenem induction. Specific activity was measured with nitrocefin (100 µM) as the substrate.

These results suggested that bla_OXA-22 and bla_OXA-60 gene expression was not repressed and that induction required an upstream-located DNA sequence, probably binding a positive regulator.

Sequence analysis of the promoter regions of the three -lactamase genes cepH, ampH and imiH, encoding CepH (cephalosporinase), AmpH (oxacillinases) and ImiH (carbapenemase), from A. hydrophila revealed sequence similarities (TTCAC motifs) that have been proposed to be consensus sequences for binding of regulators such as the BlrA protein (Avison et al., 2004). Comparison of the bla_OXA-22 and bla_OXA-60 upstream sequences revealed a highly homologous region of 17 bp including a tandem repeat of (GTTAC-n₄-GTTAC) similar to the ‘cre/blr-tag’ (TTCAC-n₆-TTCAC) from Aeromonas sp. (Avison et al., 2004). These tandem repeats were located at position –62 to –49 relative to bla_OXA-22 (+1) and at position –80 to –67 relative to bla_OXA-60 (+1) (Fig. 2). In addition, a third copy of this GTTAC motif was found further upstream of the bla_OXA-22 (+1), at position –188 to –184 (data not shown). However, these repeats were not involved in the regulation of bla_OXA-22 and bla_OXA-60 since: (i) the region essential for induction lies between –240 and –159, while the GTTAC-n₄-GTTAC repeat lies at –80 bp, and (ii) deletion of the tandem repeat GTTAC-n₄-GTTAC upstream of the chromosomal copy of the bla_OXA-22 gene did not significantly change the induction properties of OXA-22.

Site-directed mutagenesis of –35 promoter sequences
In order to determine the involvement of the –35 promoter sequences CTGCAG and TGGCCG, respectively, in the expression of the bla_OXA-22 and bla_OXA-60 genes, this sequence was replaced by the E. coli ⁷⁰ –35 promoter-consensus sequence TTGACA (pMR5, pMR10) (Fig. 3). Increased expression of both bla_OXA-22 and bla_OXA-60 was obtained in, respectively, R. pickettii PIC-1OXA-60 and R. pickettii PIC-1OXA-22 with the E. coli ⁷⁰ –35 promoter-consensus sequence (Fig. 3b, c). The level of expression was comparable to that obtained upon induction (Fig. 3b, c). These results suggested that the weak basal expression of the bla_OXA genes was the result of a weak activity of their promoters, due to inefficient –35 promoter sequences, and that their increased expression upon induction relies on the binding of an activator to the upper DNA sequence, which may contribute to the stabilization of the RNA polymerase on the promoter sequences.

Taken together, these results indicate that both -lactamases are not repressed in the absence of inducer and are positively regulated upon induction. These findings argued for the presence of a specific activator-binding sequence near the promoter of each -lactamase gene as for Aeromonas spp. (Avison et al., 2004).

Genetic environment of bla_OXA-22 and bla_OXA-60
Since genes encoding a bacterial transcription factor are often linked to one of the regulated genes (Niumsup et al., 2003), the sequences upstream and downstream of the bla_OXA-22 and bla_OXA-60 genes were further examined. Analysis of the DNA sequence revealed several ORFs. ORF-D, which encoded a putative protein of 198 aa that shared no significant identity with known protein sequences available in the GenBank database, was found 229 bp upstream of the bla_OXA-22 gene. Downstream of it, the 3' end of another ORF was identified, ORF-E, which encoded a protein that shared 35 % amino acid identity with a putative transcription activator of the LysR family from Streptomyces coelicolor (Redenbach et al., 1996). Its function remains unknown in R. pickettii. Several ORFs were identified in the DNA sequences surrounding the bla_OXA-60 gene that shared sequence identity with chromosomally encoded genes of Ralstonia solanacearum (Salanoubat et al., 2002) and Chromobacterium violaceum (Brazilian National Genome Project Consortium, 2003; Girlich et al., 2004b), but their function remains unknown in R. pickettii. In order to test whether any of the proteins encoded in these ORFs might be involved in the regulation of expression of the oxacillinases in R. pickettii, these ORFs were knocked out. R. pickettii PIC-1ORF-D, PIC-1ORF-E, PIC-1ORF-RP2, PIC-1ORF-RP3 and PIC-1ORF-RP4 were analysed for -lactamase expression and induction (Table 3). Among these R. pickettii PIC-1 isogenic mutants, PIC-1ORF-RP3 was the only one for which a modification of -lactamase expression was observed (Table 3). The R. pickettii PIC-1ORF-RP3 strain presented a single copy of pORF-RP3 integrated into ORF-RP3, thus disrupting this ORF. The R. pickettii PIC-1ORF-RP3 strain was more susceptible to all -lactams than wild-type R. pickettii PIC-1 although it expressed a sixfold higher constitutive -lactamase level than that of the parental strain (Table 3). Furthermore, R. pickettii PIC-1ORF-RP3 lacked inducible -lactamase expression, thus indicating that ORF-RP3 encoded a protein probably involved in -lactamase expression (Table 3). Trans-complementation of R. pickettii PIC-1ORF-RP3 with recombinant plasmid pLAF-RP3, expressing ORF-RP3 from a multicopy plasmid pLAFR3 (Staskawicz et al., 1987), resulted in recovery of a wild-type -lactam susceptibility profile and an inducible -lactamase expression in R. pickettii, although at a lower level (Table 3). Both -lactamase genes were inducible, as revealed by IEF results (data not shown). These data confirmed that the observed phenotype of mutant R. pickettii PIC-1ORF-RP3 strain was linked to inactivation of the ORF-RP3. Partial complementation probably resulted from non-optimal expression of ORF-RP3 from the plasmid pLAF-RP3. Interestingly, ORF-RP3 and bla_OXA-60 shared no sequence identity with any other ORFs on the chromosome of R. solanacearum, which is the species most related to R. pickettii. Furthermore, as indicated previously (Girlich et al., 2004b), these two genes seem to have integrated together between two genes sharing high nucleotide identity with two contiguous genes located in the R. solanacearum genome (Salanoubat et al., 2002). R. solanacearum contains a single oxacillinase gene that shared 63 % nucleotide identity with bla_OXA-22. The expression of this oxacillinase gene is not inducible (data not shown) and it is tempting to argue that the absence of an ORF-RP3 in R. solanacearum might explain the absence of -lactamase induction in that species.

View this table: [in this window] [in a new window]   Table 3. -Lactamase activity of R. pickettii PIC-1 and isogenic strains, deficient in ORFs surrounding both blaOXA genes and that of R. pickettii PIC-1ORF-RP3, deficient in RP3, before and after transformation with a plasmid carrying ORF-RP3

Environmental stress response of mutant R. pickettii PIC-1ORF-RP3
Nishino et al. (2003) demonstrated that the response regulator EvgA controlled the expression of multiple genes conferring antibiotic resistance in E. coli by regulating the expression of drug transporters. Ramos-Aires et al. (2004) showed that inactivation of the GlmR transcriptional regulator, involved in amino sugar metabolism, dramatically sensitized P. aeruginosa to a large variety of antibiotics, suggesting interaction between several regulatory pathways. Similarly, R. pickettii PIC-1ORF-RP3, deficient in RP3, was more susceptible to all -lactams than wild-type R. pickettii PIC-1 (data not shown) and the wild phenotype could be restored when ORF-RP3 was expressed in trans from plasmid pLAF-RP3. In order to understand the physiological role of the RP3 protein, we attempted to further characterize the R. pickettii PIC-1ORF-RP3 strain. Changes in the morphology of the bacteria could be observed on Gram staining: ORF-RP3 deletion mutants were thinner, longer and remained in chains. The growth rate and the ability to resist drastic changes in temperature, pH and osmolarity were also examined. The growth rates of R. pickettii PIC-1 and of R. pickettii PIC-1ORF-RP3 strains at 37 °C in TS broth were similar except for a longer lag phase for the mutant strain (Fig. 4). Stationary-phase cultures of both strains were exposed to a sudden temperature shift from 37 to 50 °C. After 15 min at 50 °C, the mortality of the R. pickettii PIC-1ORF-RP3 culture was about 30 times higher than that observed for the parental culture (Fig. 4). Exposure of mutant R. pickettii PIC-1ORF-RP3 to 2 M NaCl resulted in a considerable decrease in the number of viable cells (6 % survival) relative to that in the wild-type (100 % survival). Thus, these results suggested that inactivation of ORF-RP3 seriously impairs the capacity of R. pickettii PIC-1ORF-RP3 to adapt rapidly to both temperature and osmotic variations, similarly to what was observed by Ramos-Aires et al. (2004) upon inactivation of the glmR gene in P. aeruginosa. Our results were also in agreement with those of Nishino et al. (2003), who showed that evgA-overexpressing E. coli strains had an increased resistance to high ionic strength. Indeed, overexpression of this gene resulted in a better ability to survive at low pH and high osmolarity. By contrast with this system, exposure to an acidic pH of R. pickettii PIC-1 and of its isogenic mutant did not reveal any modifications in their antibiotic resistance patterns. In both cases no cells survived upon 1 h exposure to pH 2.0 and 100 % survived upon 2 h exposure to pH 5.2 (data not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 4. Growth at 37 °C and resistance to heat shock of the R. pickettii PIC-1 parental strain () and isogenic strain PIC-1ORF-RP3 (). (a) Growth curve in TS medium at 37 °C. (b) Cell survival assay after exposure to heat shock at 50 °C.

Alteration of ORF-RP3 in R. pickettii results in a complex, pleiotropic phenotype, probably resulting from perturbation of the peptidoglycan structure in R. pickettii PIC-1ORF-RP3 mutant, as evidenced by its abnormal response to temperature and osmotic stress. Further studies of the cell shape and of the amount of LPS and phospholipids will be needed to confirm this hypothesis. It is reasonable to think that RP3, like BlaRI from B. licheniformis, could be involved in a system of signal transduction through the membrane (Kobayashi et al., 1987).

	ACKNOWLEDGEMENTS

 
We thank S. Génin for providing plasmid pLAFR3. This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA 3539), Université Paris XI, Paris, France, and mostly by the European Community (6th PCRD, LSHM-CT-2003-503-335).

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