a Laboratoire de BactériologieVirologie, Faculté de Pharmacie, 1 rue Gaston Veil, 44035 Nantes; b Laboratoire de Bactériologie, Virologie, Hygiène hospitalière, CHU, Nantes, France
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Abstract |
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Introduction |
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Numerous mutations have been described in the ampC promoter of clinical strains hyperproducing a chromosomal cephalosporinase,47 but the precise role of these mutations has not been determined. Some of them may be simple polymorphisms while others are certainly responsible for an increased trancription rate.
The most frequently described E. coli ampC strong promoter harbours mutations at positions 88, 82, 42, 18, 1 and +58.5 The 42CT transition creates a new displaced 35 perfect consensus sequence, TTGACA, separated by 17 bp from a new 10 sequence, caused by the 18G
A transition.7 Less frequent mutations have been described in clinical strains, particularly in the attenuator and in the fourth base of the 35 box. This last mutation has also been described in mutants selected in vitro.8
In this paper, different ampC promoters from E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (cat) gene in the reporter plasmid pKK232-8.9 By site-directed mutagenesis, we have investigated the role of the 32 and 42 mutations that create a consensus TTGACA sequence.
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Materials and methods |
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All plasmids were obtained by transforming highly competent JM109 cells (Promega, Charbonnières, France). pGEM-T Easy vector (Promega) was used for post-PCR cloning experiments, pKK232-8 (Amersham Pharmacia Biotech, Uppsala, Sweden) as a promoterless reporter gene and pALTER-Ex2 (Promega) for site-directed mutagenesis experiments. Strain 96006296 is a susceptible E. coli clinical isolate. E. coli strains 96004153 and 96010266 are AmpC hyperproducers.6 Details of the plasmids used in this study are given in Table I.
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Primers AB1 (151GATCGTTCTGCCGCTGTG134) (5' to 3') and ampC2 (120GGGCAGCAAATGTGGAGCAA101) (5' to 3') were used to amplify a 271 bp fragment containing the 35 box, the 10 box and the attenuator from the E. coli ampC promoter.10 PCR amplification was performed in a PerkinElmer 480 DNA thermal cycler (PerkinElmer Applied Biosystems, Cergy-Pontoise, France). PCR reactions were performed in a final volume of 50 µL containing 10 mM TrisHCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM of each nucleotide, 0.5 µM of each primer, 2.5 units of Taq DNA polymerase (Gibco BRL Life Technologies SARL, Cergy Pontoise, France) and 10 ng of target DNA. After 90 s denaturation at 94°C, 30 PCR cycles were performed, each consisting of 90 s denaturation at 94°C, 30 s annealing at 57°C and 60 s extension at 72°C; a final extension step of 10 min at 72°C was performed.
Construction of reporter plasmids
The 271 bp PCR fragment was cloned in the post-PCR cloning vector, pGEM-T Easy. This plasmid was then digested with EcoRI and the fragment containing the ampC promoter was recovered from a low-melting-point agarose gel, blunt ended with the Klenow fragment of E. coli DNA polymerase I, and ligated into the SmaI restriction site of pKK232-8, previously treated with calf intestinal phosphatase (Promega). After overnight incubation at 16°C, these ligation products were used to transform highly competent JM109 cells (Promega).
Preparation of mutant promoters by site-directed mutagenesis
Site-directed mutagenesis was performed using the Altered Sites II Ex2 in vitro mutagenesis system (Promega). The EcoRI-digested fragment from pGEM-T Easy was subcloned in the EcoRI site of the mutagenesis vector pALTER-Ex2. This vector contains genes for tetracycline and chloramphenicol resistance, but the latter has been inactivated. During the mutagenesis reaction, a chloramphenicol repair oligonucleotide was annealed to alkali-denatured DNA at the same time as the mutagenesis oligonucleotide, and restored resistance to chloramphenicol. The mutagenesis reaction was carried out according to the manufacturer's recommendations; mutants were selected on LB agar containing chloramphenicol 20 mg/L. The mutagenic oligonucleotides, Mut32 (41P-TGACAGTTGTCACGCTGAT23) (5' to 3') for strain 96004153 and Mut42 (51P-GCTGCTATCCTGACAGTTG33 (5' to 3') for strain 96010266 were centred on the desired mutations and 5'-phosphorylated (Genosys Biotechnologies, London, UK). The EcoRI fragments from the mutated promoters were then subcloned in pKK232-8, as previously described.
Verification of the constructions
In order to control the sequence of the inserted fragments, two primers from pKK232-8, pKK1 (122TGCGAAGCAACGGCCCGG139) and pKK2 (212AAGCTTGGCTGCAGGTCGA194) (5' to 3') were synthesized to amplify a 389 bp fragment containing the 271 bp from the ampC gene. The PCR products were then purified on a Bio-Gel P100 (Bio-Rad SA, Ivry Sur Seine, France) and sequenced in both strands with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer). Sequence analysis was performed on a ABI 373 DNA sequencer (PerkinElmer).
CAT assays
Bacteria containing each plasmid were cultured overnight in 20 mL of Luria broth. The bacterial pellet was obtained by centrifugation of the previous culture for 30 min at 10000g. After washing and resuspension in distilled water, bacteria were sonicated in a Branson Sonifier 250 (intermittent exposure: 5 x 30 s). After centrifugation, the supernatant was used for CAT determination. Total protein concentration was determined by the ESL protein assay (Boehringer Mannheim, Meylan, France) and the volume of extract used for CAT determination was adjusted to equalize the protein concentration. CAT enzyme was measured by a sandwich ELISA test performed in a microtitre plate (Boehringer Mannheim). This test, which quantifies CAT in transfected eukaryotic cells, was used according to the manufacturer's recommendation except that five dilutions of total protein (250, 25, 2.5, 0.25 and 0.025 µg/mL) were tested for each sample to be in the linear range of the test. Each determination was performed in triplicate. Untransfected JM109 cells were used as a control.
MIC determinations
The chloramphenicol MIC was determined by serial two-fold dilution in MuellerHinton agar (Difco Laboratories, Detroit, MI, USA). Inocula of 104 cfu per spot from an 18 h culture in MuellerHinton broth were applied with a Steers multiple-inoculum replicator. After 18 h incubation at 37°C, the chloramphenicol MIC was defined as the lowest concentration that prevented visible growth of the spot on the plate.
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Results |
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A 271 bp fragment containing the promoter of the gene for three different AmpC ß-lactamases from three E. coli clinical strains was cloned upstream of the cat gene of the reporter plasmid pKK232-8. The sequences of the cloned fragments are shown in the Figure. One of the promoters came from a susceptible E. coli strain (96006296); the other two were from strains hyperproducing a chromosomal cephalosporinase. These constructs were used to transform E. coli JM109 competent cells. CAT determination was then performed in two ways: (i) indirectly, by measuring the chloramphenicol MIC; and (ii) directly, using an immunoenzymatic method (Table II
).
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Role of the 42 and 32 point mutations
Using site-directed mutagenesis, the promoter from strain 96010266 was modified by reverting the CT transition at position 42. This single change in the promoter sequence resulted in the CAT concentration and chloramphenicol MIC decreasing to levels found with the promoter of a susceptible isolate (Table III
).
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Discussion |
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With this method, CAT concentration was 70- to 120-fold higher when a strong promoter was cloned than when a weak promoter from a sensitive isolate was used. At the same time, the chloramphenicol MIC increased by 64-fold. Aramori & Kojo showed a 23- to 70-fold increase in CAT activity for ampC promoters from E. coli hyperproducing a chromosomal cephalosporinase, but the sequence of these promoters was unknown.17 Recently, using a luciferase reporter gene method, Nelson & Elisha detected an eight- to 18-fold increase in promoter strength from AmpC hyperproducers.7
In 1983, by analysis of 168 promoter regions of E. coli genes, Hawley & McClure confirmed the presence of two conserved regions, the 35 hexamer and the 10 hexamer, also called the Pribnow box.18 It is now known that these elements are crucial for fixation of the subunit of RNA polymerase.19 For most promoters, there is a good correlation between promoter strength and the degree of homology to the consensus sequence for the 35 box, TTGACA, and for the 10 box, TATAAT. The distance between these two sequences also plays an important role in the strength of the promoter, the optimal distance being 17 bp.20 In E. coli wild-type ampC promoter, a 35 TTGTCA sequence is separated by 16 bp from a 10 TACAAT sequence. The most frequently described strong ampC E. coli promoter presents a C
T transition at base 42 that creates a perfect TTGACA box upstream of the normal 35 box, modifying the transcription initiation site.4 By site-directed mutagenesis, we confirmed that the abolition of this single mutation in the promoter from strain 96010266 decreased its strength to the level of a sensitive isolate. This does not mean that other mutations present in this promoter do not play any role. The 18 mutation, by creating a new 10 box, certainly plays an important role.
In the ampC promoter from strain 96004153, the TA transversion at position 32 gave perfect homology to the 35 sequence and was suspected to increase the transcription rate. This hypothesis was confirmed by reverting this mutation using site-directed mutagenesis; the strength of the mutated promoter was decreased by about 13-fold. However, it was still stronger than the weak promoters (96006296), suggesting that at least one of the other mutations present in this strong promoter must be implicated in the increased transcription rate. Among the other mutations, one (+24 mutation) is localized in the attenuator. This structure has been identified in the E. coli ampC promoter and is involved in low-level transcription of the gene.21 A mutation in this part of the gene could destabilize the hairpin structure and increase transcription. We tried to revert the mutation at position +24, but were not successful, probably because of the hairpin structure.22
With this system, it could be interesting to determine the role played by mutations in other parts of the promoter, particularly in the Pribnow box, since such mutations have been described in laboratory mutants but, as yet, not in clinical strains.8
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Acknowledgments |
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Notes |
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References |
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Received 30 September 1999; returned 21 December 1999; revised 12 January 2000; accepted 21 January 2000