Expression of the aac(6′)-Ib-cr Gene in Class 1 Integrons

TEXT

Plasmid-mediated quinolone resistance (PMQR) was extensively characterized in the last decade. The aminoglycoside 6′-N-acetyltransferase type Ib-cr [AAC(6′)-Ib-cr] protein is a PMQR determinant and a variant of the aminoglycoside resistance enzyme AAC(6′)-Ib with an extended acetylation spectrum conferring low-level resistance to ciprofloxacin and norfloxacin (1, 2). The aac(6′)-Ib-cr gene has been described in Gram-negative bacteria (mainly Enterobacteriaceae) and is usually located within a gene cassette contained in a class 1 integron (3–5). Integrons are genetic platforms that mobilize usually promoterless gene cassettes and express their genes from a common promoter named P_C (6, 7). Several P_C variants of different strengths have been described in class 1 integrons, including weak and strong variants (8). The aac(6′)-Ib-cr gene cassette is usually located at the first position in class 1 integrons, being associated with a weak (P_CW or P_CH1) P_C variant. A 101-bp sequence referred to as qacE¹⁰¹ is always found upstream of the aac(6′)-Ib-cr gene cassette (6, 7) (Fig. 1). This fragment has been described upstream of other genes [aac(3)-Via] or gene cassettes (aadA16 and aacA29) and is identical to the promoter region of the qacEΔ1 and sul1 genes, which are found at the 3′ end of >90% of class 1 integrons (9–12). In silico and experimental studies showed the presence of a functional promoter in qacE¹⁰¹ (7, 9–12). Pérez-Moreno et al. (4) suggested that the qacE¹⁰¹ promoter together with the integron P_C promoter may be involved in aac(6′)-Ib-cr expression, but this has not been shown experimentally. To elucidate the respective contribution of the P_C and qacE¹⁰¹ (named P_A) promoters in the expression of aac(6′)-Ib-cr, we constructed P_CW-P_A-lacZ transcriptional fusions (see Table S1 in the supplemental material), with wild-type or mutated versions of both promoters, and quantified the resulting β-galactosidase activities. The P_CW-P_A region was amplified from the Klebsiella pneumoniae C1911 strain that contains a class 1 integron with aac(6′)-Ib-cr as the first gene cassette (4) (see Table S2 in the supplemental material). The weak P_CW promoter was chosen because it is the most common P_C variant in the aac(6′)-Ib-cr-containing integrons, according to our survey of online integron sequences (see Table S3 in the supplemental material).

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FIG 1

Partial nucleotide sequence (1,500 bp) of the class 1 integron of the K. pneumoniae C1911 strain (GU906294 ). The 101-bp region is highlighted in gray; the −35 and −10 promoter sequences, putative Shine-Dalgarno, 3 putative start codons, and stop codon are indicated in bold and underlined. TSS is indicated by a triangle. Putative R′ motifs of aac(6′)-Ib-cr and aac(6′)-Ib cassettes are boxed. The 30-bp additional sequence in the aac(6′)-Ib-cr cassette is indicated in italics. Cloned fragments of 480, 247, and 639 bp correspond to sequences between nucleotides 299 and 778, 532 and 778, and 653 and 1291, respectively. Nucleotides are numbered as for GenBank accession number GU906294 . The amino acids of identified peptides by NanoLC-MS/MS are in boldface.

The β-galactosidase activity drastically fell when the P_A (pP_CWP_A*) or both promoters (pP_CW*P_A*) were mutated (Fig. 2A). Besides, mutation (P_CW*P_A) or deletion (pP_A) of P_CW reduced the overall level of expression by only 15% (Fig. 2A). Relative quantification of lacZ transcripts by reverse transcriptase quantitative PCR (RT-qPCR) was performed using the 2^−ΔΔCt method (13). lacZ copy numbers were normalized to the endogenous dxs gene and compared to the control condition (pSU38ΔtotlacZ control). Transcripts quantification showed similar trends (Fig. 2B). Altogether, these results indicate that expression of aac(6′)-Ib-cr in integrons is mostly under the control of the P_A promoter, whereas P_CW makes only a minor contribution. Actually, it seems that the P_A promoter allows compensation for the weakness of the integron P_C promoter. The qacE¹⁰¹ fragment therefore consists of an expression pseudocassette for downstream gene cassettes. Some other expression pseudocassettes were reported to drive the expression of the bla_OXA-10 and aadA1 gene cassettes in integrons (14). Because of an incomplete attC site, the mobility of these pseudocassettes remains uncertain. The pseudocassettes may represent an alternative strategy to counterbalance the decrease in expression level along the integron cassette arrays and/or the weakness of the P_C promoter. As shown for In53, it might also favor an increase in the number of expressed gene cassettes in multiresistant integrons (16).

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FIG 2

(A) β-Galatosidase activity; results for DH5α Escherichia coli strains carrying the pSU38ΔtotlacZ control (C), pP_CWP_A (1), pP_CW*P_A (2), pP_CWP_A* (3), pP_CW*P_A* (4), and pP_A (5) plasmids. (B) Expression ratio of lacZ transcripts for all strains compared with the control strain (C). All P values for strain comparisons are <0.05, except for 2 versus 5 (A) and 1 versus 2 (B).

In addition to the presence of the qacE¹⁰¹ fragment, the aac(6′)-Ib-cr gene cassette differs from the aac(6′)-Ib gene cassette by an additional 30-bp fragment in 5′ between the qacE¹⁰¹ fragment and the beginning of the aac(6′)-Ib gene cassette (Fig. 1). This sequence exhibits an additional in-frame ATG start codon located 22 bp downstream of the transcription start site (TSS) determined by 5′ rapid amplification of cDNA end (RACE) experiments (Fig. 1) (15). This start codon is preceded by a conserved Shine-Dalgarno sequence, suggesting that the encoded AAC(6′)-Ib-cr protein could be longer than the AAC(6′)-Ib protein (Fig. 1). To identify the initiation codon of the AAC(6′)-Ib-cr protein, we purified a histidine C-terminal-tagged AAC(6′)-Ib-cr protein that was digested into trypsic peptides for nanoscale liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis (Fig. S1 in the supplemental material). The presence of the AAC(6′)-Ib-cr protein was confirmed, with a coverage of 51%, through identification of 10 distinct peptides (see Fig. S2 in the supplemental material). Note that the peptide YSIVTNSNDSVTLR, identified 13 times, contained the valine (underlined) encoded by the usual GTG start codon for AAC(6′)-Ib protein translation (Fig. 1).

This clearly shows that the GTG triplet is not the start codon of the protein and suggests that the upstream in-frame ATG is the start codon; AAC(6′)-Ib-cr is thus probably 199 amino acids (aa) long. This start codon position is in agreement with the position of the P_A promoter characterized here and with earlier structural studies of this protein, in which the cloned and overexpressed gene was the 199-aa encoding form (1). It also supports the evolutionary model of AAC(6′)-I proteins that go through substitutions impacting the antibiotic acetylation profile and through variability in the N-terminal domain (16–18). The biological implications of the N-terminal variability of AAC(6′)-I proteins remain to be explored.