Transcriptional Analysis of the bla_CTX-M-2 Gene in Salmonella enterica Serovar Infantis

Abstract

Transcriptional organization of bla_CTX-M-2 present in a multiresistance plasmid of Salmonella enterica serovar Infantis suggests the presence of more than one promoter involved in the expression of the β-lactamase gene. At least two bla_CTX-M-2-specific mRNAs (near to 1 kb and 5 kb) were evidenced. Two +1 signals were detected at −22 bp and −59 bp of bla_CTX-M-2 defining two putative promoters.

CTX-M enzymes represent a growing family of class A extended-spectrum β-lactamases (ESBLs) that preferentially hydrolyze cefotaxime over ceftazidime, both oxyimino cephalosporins, including more than 30 members organized in six groups (3). In Argentina, the more prevalent ESBL is CTX-M-2 (20, 22, 23), representative of the CTX-M-2 group (3).

Several reports have identified −35 and −10 promoter regions included in an ISEcp1-like element upstream to bla_CTX-M-13 and bla_CTX-M-14 (6), bla_CTX-M-15 (2, 13), bla_CTX-M-17 (4), bla_CTX-M-19 (18), bla_CTX-M-21 (accession number AJ416346), bla_CTX-M-25 and bla_CTX-M-26 (15), bla_CTX-M-32 (5) and other bla genes encoding CTX-M enzymes (8, 26), involved in the expression of these enzymes.

By contrast, an open reading frame (orf513) associated to unusual class 1 integrons was found upstream to bla_CTX-M-2 and bla_CTX-M-9 (1, 7, 14, 19, 25, 28). Even if these genes have been suggested to be associated also to ISEcp1 (3), this association has not been confirmed up to now. To our knowledge no data have been reported related to the expression of these enzymes.

In this work we studied the transcriptional organization of bla_CTX-M-2 from a Salmonella enterica serovar Infantis plasmid. Plasmid pS21 is a conjugative plasmid from a cefotaxime-resistant Salmonella serovar Infantis clinical isolate (S21). pS21 carries InS21, an unusual class 1 integron with the bla_CTX-M-2 gene associated to orf513 (7).

Expression of bla_CTX-M-2 in the presence of its upstream region.

A fragment of 1.5 kb (including bla_CTX-M-2 plus a 500-bp upstream region), obtained by PCR with Bla2 (5′-GATACCTCGCTCCATTTATTGC) and Bla-down (5′-TTGACTGTCGACCCCAAATCC) from pS21, was cloned into the pCR2.1 vector (Invitrogen), to obtain recombinant plasmid pCRC. The insert of pCRC was then removed by digestion with EcoRI and subcloned into the plasmid vector pPR328 (21). Two recombinant plasmids (pPR328C5 and pPR328C6) with the insert in both orientations were confirmed by sequencing. Both recombinants, regardless of the insert orientation, were resistant to cefotaxime, ampicillin, piperacillin, cephalothin, and aztreonam. Susceptibility was restored in the presence of clavulanic acid (data not shown). After isoelectrofocusing of crude extracts, a single band (pI 8.2) of equivalent intensity with either recombinant could be detected using an iodometric system (with ampicillin [500 μg/ml] or ceftriaxone [1,000 μg/ml] as substrates) (24). Results of this experiment suggested that at least one promoter sequence is present in the 500-bp region upstream to bla_CTX-M-2.

Sequence analysis of the regions flanking bla_CTX-M-2.

Sequence analysis of the 500-bp region upstream of bla_CTX-M-2 in InS21 reveals two different regions: (i) a sequence of 266 bp 96% identical to the upstream region of the KluA-1-encoding gene (a chromosomal β-lactamase from Kluyvera ascorbata; EMBL accession number AJ272538), with identical putative ribosome-binding site sequences (AGAGG) and −10 promoter region (TTGAAG) to those described for other enzymes of same group, but lacking a −35 promoter region (9, 10, 12); and (ii) a distal segment which is 100% identical to the 234-bp common region (CR) downstream of orf513 (CR1) described in unusual class 1 integrons (11) including the right-hand boundary of the CRs (17). A putative promoter in this region (similar to the Escherichia coli consensus promoter) has been proposed for dfrA10 of In7 (16) and could function as another promoter for bla_CTX-M-2 expression (Fig. 1A). A 28-bp inverted-repeat region at 6 bp downstream to the bla_CTX-M-2 stop codon could operate as a factor-independent transcription terminator (Fig. 1B).

FIG. 1.

Surrounding sequences of bla_CTX-M-2 gene and primer extension analysis. A) Sequence analysis of the 500-bp upstream bla_CTX-M-2 region. The ATG start codon and the ribosome-binding site are indicated in boldface. The sequence with homology to the upstream region of bla_KluA-1 is underlined with a solid line; the −35 and −10 sequences of the P1 promoter are boxed with solid lines; the −35 and −10 sequences of the P2 promoter are boxed with dotted lines. The rest of the sequence corresponds to the 3′ end of the CR1 element; the −10 promoter sequence proposed for bla_CTX-M-2-type by Saladin et al. is boxed in dotted lines; the −35 and −10 promoter regions proposed for dfrA10 of In7 are underlined with dotted lines. B) bla_CTX-M-2 downstream region. In boldface is indicated the TGA bla_CTX-M-2 stop codon; the putative transcription terminator is underlined. C) Primer elongation products (previously treated with ribonuclease) were analyzed by electrophoresis on 6% denaturing polyacrylamide gels using an ALF Express sequencer (Pharmacia). Lane 1, primer elongation products obtained from total RNA of S21 using the Bla-up oligonucleotide. Distances (bp) from the start codon of bla_CTX-M-2 are indicated with each arrowhead. Lanes T, G, C, and A, sequencing reactions used as marker. The +1 transcriptional starting sites (−22 and −59 bp) of bla_CTX-M-2 are indicated in boldface.

Transcriptional characterization of bla_CTX-M-2.

Total RNA was extracted using the FastRNA kit BLUE (BIO 101) according to the manufacturer's recommendations. Samples were dissolved in 0.2% diethyl pyrocarbonate-treated water. The probe used was a 902-bp PCR amplicon produced from pS21 DNA with the Bla1 and Bla2 primers (7), labeled by random primer synthesis employing [α-³²P]dCTP (27). Hybridization was performed under high-stringency conditions with an incubation temperature of 42°C for 16 h. At least two bla_CTX-M-2 transcripts were detected by Northern blot in S21 (Fig. 2). Considering bla_CTX-M-2 gene size, the 0.95- to 1.38-kb bla_CTX-M-2-specific transcripts may correspond to monocistronic transcripts. However, larger (5-kb) bla_CTX-M-2-positive transcripts were also detected, which may correspond to polycistronic mRNAs.

FIG. 2.

Northern blot. A) RNA (5 to 10 μg) was electrophoresed on a formaldehyde-containing 1.2% agarose gel in MOPS (morpholinepropanesulfonic acid) running buffer. The presence of 23S and 16S rRNA bands showed the integrity of mRNA. B) Autoradiograph where the bla_CTX-M-2-specific positive transcripts are detected. M, RNA marker, 0.28 to 6.58 kb (Promega).

The organization of bla_CTX-M-2-specific mRNA was explored by reverse transcription-PCR (RT-PCR) strategies (Fig. 3). RNA (3 μg) was reverse transcribed in a final volume of 100 μl using 2 μl hexanucleotide (Promega) or 10 μl reverse primer, 10 μM (Bla2 or Bla-up), according to the Reverse Transcription System (Promega). Ten microliters of RT product was used for specific amplification by PCR experiments in a final volume of 100 μl. RNA samples without RT were used as negative controls. Each RT-PCR was performed in triplicate. Every PCR (I, II, III, and IV) done on S21 RNA subjected to random RT (with hexanucleotides) and directed RT (with Bla2 primer) was positive, and the predicted amplicons were obtained in each case. These results and the presence of bla_CTX-M-2-positive transcripts of ∼5 kb suggest that at least one promoter located in CR1 (or even upstream of CR1) could mediate bla_CTX-M-2 expression.

FIG. 3.

RT-PCR strategies for bla_CTX-M-2 transcript analysis. A) Directed RTs with Bla2 or Bla-up primers and random RT with hexanucleotides. B) PCR amplifications follow each RT. Over the dotted lines, the expected amplicon sizes (kb) are shown. AUG, start codon of bla_CTX-M-2 (heavy black line); CR1, 3′ end of common region of In6/In7 (heavy grey line). Primers employed in the directed RTs and PCRs are designated with cardinal numbers (1, Bla1; 2, Bla2; 3, Bla-up; 4, Bla-down) (7).

Primer extension was done using the 5′-labeled Bla-up oligonucleotide (Cy5 ALF Express; Amersham Pharmacia Biotech) complementary to a sequence 97 to 117 bp downstream from the initiation site of the bla_CTX-M-2 gene. The extension was performed as described for RT employing 30 μg of RNA and 2 μl of labeled primer (50 pmol/μl). Two +1 signal transcriptional starting sites were identified (Fig. 1C). One of them was located at −22 bp from the start codon of bla_CTX-M-2 gene, and the deduced −35 (ACTTTT) and −10 (TTGAAG) boxes of the putative bla_CTX-M-2 promoter (P1) were separated by 18 bp. A second transcriptional starting site (−59 bp from the start codon) allowed the proposal of a second putative promoter (P2) with CAGGCT and AGGTTT as −35 and −10 boxes, even if, according to the relative signal intensities of cDNA bands obtained, P1 may be stronger than P2. These two putative promoters defined in this upstream region (P1 and P2) could account for monocistronic transcripts. Based on these data, the −10 region of the P1 promoter sequence was identical to those described by other authors (9, 10, 12). However, the -35 region has not been previously described in any other promoter search (to the best of our knowledge).

Promoter prediction on the 500-bp sequence upstream of bla_CTX-M-2 was carried out by Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html). Three sequences were detected by this program as putative promoters with a score cutoff at 0.90, all located proximal to the start codon of bla_CTX-M-2. One of them included the −10 and −35 regions of the proposed P1 promoter, and the other two sequences contained only the −10 region of the P1 and P2 putative promoters.

Although several putative promoters have been proposed for bla_CTX-M-2 group genes by sequence analysis, this is the first experimental characterization of two transcriptional starting sites that support the promoter prediction program results. Putative −35 and −10 promoter sequences were proposed by Saladin et al. (26) in two Proteus mirabilis isolates producing CTX-M-2-type β-lactamases. They employed an internal ISEcp1 forward primer (ISEcp1 U1) containing the typical −35 promoter region, for PCR amplification. However, immediately downstream to this primer, a 17-bp sequence identical to the 3′ end of CR1 (where is included orf513) was described. Considering the 3′ end sequence of CR1, a nonspecific hybridization of the ISEcp1 U1 primer could not be disregarded. The 266-bp sequence present immediately upstream to bla_CTX-M-2-type genes in those isolates (where no promoter sequences were searched for [26]) is identical to that analyzed here.