ABSTRACT
Expression of the quinolone resistance gene qnrS1 is increased by quinolones, but unlike induction of some other qnr genes, the bacterial SOS system is not involved and no lexA box is found upstream. Nonetheless, at least 205 bp of upstream sequence is required for induction to take place. An upstream sequence bound to beads trapped potential binding proteins from cell extracts that were identified by mass spectrometry as Dps, Fis, Ihf, Lrp, CysB, and YjhU. To further elucidate their role, a reporter plasmid linking the qnrS1 upstream sequence to lacZ was introduced into cells of the Keio collection with single-gene deletions and screened for lacZ expression. Mutants in ihfA and ihfB had decreased lacZ induction, while induction in a cysB mutant was increased and dps, fis, lrp, yjhU, and other mutants showed no change. The essential upstream sequence contains potential binding sites for Ihf and DnaA. A dnaA deletion could not be tested because it provides essential functions in cell replication; however, increased dnaA expression decreased qnrS1 induction while decreased dnaA expression enhanced it, implying a role for DnaA as a repressor. In a mobility shift assay, purified IhfA, IhfB, and DnaA proteins (but not CysB) were shown to bind to the upstream segment. Induction decreased in a gyrA quinolone-resistant mutant, indicating that GyrA also has a role. Thus, quinolones acting through proteins DnaA, GyrA, IhfA, and IhfB regulate expression of qnrS1.
INTRODUCTION
Among the mechanisms of plasmid-mediated quinolone resistance, Qnr proteins are the most diverse group, with members distributed widely in enteric Gram-negative bacteria (1–3). Qnr proteins belong to the pentapeptide repeat protein family. They protect DNA gyrase and DNA topoisomerase IV from inhibition by quinolones (4–6). Seven families of plasmid-mediated qnr genes, qnrA, qnrB, qnrC, qnrD, qnrE, qnrS, and qnrVC, have been identified in plasmids derived from clinical isolates (7, 8). Related qnr genes have also been found in the chromosomes of both clinical and environmental, especially aquatic, bacteria. QnrS1 in particular is similar in sequence to proteins encoded by chromosomal genes in species of Vibrio (9, 10).
Although qnr genes have been found on diverse plasmids, there are similarities in the DNA sequences flanking qnr genes in certain qnr families. qnrA and qnrB are often located in complex sul1-type integrons downstream from an insertion sequence (11, 12). qnrS genes, in contrast, are not part of integrons but usually have a flanking Tn3 transposon (13, 14). Such a conserved genetic environment of qnr genes may reflect a limited number of acquisition events of the genes from their chromosomal reservoirs during horizontal transfer to new hosts.
Fluoroquinolones such as ciprofloxacin inhibit type II topoisomerases and lead to DNA strand breaks in bacteria (15). Resulting from this DNA damage, ciprofloxacin induces the bacterial SOS system. We previously found an SOS-independent induction of qnrS1 by quinolones (16). Unlike qnrB, qnrD, and qnrE, which are preceded by a LexA box and induced by quinolones in an SOS-dependent manner (probably for qnrE based only on sequence data) (17, 18), no LexA binding site was found in the region upstream from the start codon of qnrS1 in plasmid pMG306, and induction occurred in an Escherichia coli lexA mutant (16). Therefore, we postulated that DNA sequence upstream from qnrS1 and other regulatory factors must play a key role in the SOS-independent induction of qnrS1. In this study, we identified genetic elements and regulatory proteins contributing to qnrS1 induction by ciprofloxacin.
RESULTS
Upstream sequence of qnrS1 and the relationship of qnrS1, qnrS2, and other chromosomal qnrs in Vibrio species.Putative Shine-Dalgarno and −10 and −35 promoter sequences were found upstream of qnrS1 (Fig. 1). The upstream sequence also contained potential DnaA binding sites as well as putative Ihf binding sites. The 200-bp sequences upstream from qnrS1 are strikingly conserved among qnrS1 plasmids in GenBank. The 200-bp sequences 5′ to qnrS2 are also highly conserved on plasmids and contain putative DnaA and Ihf binding sites but are markedly different from the upstream sequence of qnrS1, with only 58% sequence identity and 25% gaps, implying a separate origin for the two common QnrS varieties, which differ in 17 of 218 amino acids. Upstream sequence data for other qnrS alleles is not currently available.
DNA sequence 5′ to qnrS1 in plasmid pMG306. The qnrS1 start codon (+1 to +3) and predicted Shine-Dalgarno sequence (−10 to −5), −10 promoter (−40 to −35), and −35 promoter (−64 to −59) are in uppercase. Predicted DnaA binding sites on the positive strand (−264 to −256) and negative strand (−177 to −185, −42 to −50, and −36 to −44) are in red. Predicted IHF binding sites on the positive strand (−73 to −58) and on the negative strand (−322 to −337, −74 to −89, −50 to −65, and −45 to −60) are in blue. Overlapping sites are in green. Double-underlined deletions at −200 to −194, −194 to −189, and between −157 and −142 prevented induction, while deletion of −209 to −205 did not.
V. splendidus qnrVS1 has been considered a potential source of plasmid-carried qnrS1 based on the identity of amino acid sequences (84%) available in 2007 (19), but sequences upstream of qnrVS in V. splendidus differed substantially from the 359 bp upstream of qnrS1, with the 200 bp immediately upstream of qnrVS1 showing only 53% identity with 33% gaps relative to qnrS1 (see Fig. S1 in the supplemental material). Notably, a BLASTP search of GenBank disclosed three other Vibrio species with Qnr proteins having a higher degree of amino acid identity to QnrS1, namely, Vibrio parahaemolyticus (97% identity), Vibrio mytili (97% identity), and Vibrio ostreicida (96% identity). In these species, the 200 bp of sequence upstream of the qnr genes also had a striking 88% identity, with no gaps, to the sequence upstream of qnrS1 (Fig. S2). Thus, plasmid-carried qnrS1 appears more likely to have come more directly from V. parahaemolyticus, V. mytili, V. ostreicida, or a related Vibrio species than from V. splendidus.
Expression of qnrS1 in truncated plasmids in response to ciprofloxacin.We transformed pMG306 into E. coli DH10B (recA mutant). After ciprofloxacin exposure at half the MIC (½ MIC), a 15-fold increase in qnrS1 transcript levels was seen. This induction was similar to that in strain J53 (recA+), in which ciprofloxacin increased transcription of qnrS1 by 19-fold (Table 1). Since RecA is an essential component of the SOS response, these results confirmed that the induction of qnrS1 expression by ciprofloxacin occurs in an SOS-independent manner (16).
Relative expression level of qnrS1 in different strains and plasmids
To determine the DNA sequence upstream of qnrS1 that is required for induction, we constructed five qnrS1 recombinant plasmids, which contained the entire coding region of qnrS1 and various lengths of upstream sequence (Fig. 1). The recombinant plasmids were then transformed into E. coli DH10B, and the qnrS1 transcript levels were determined after exposure to ciprofloxacin at ½ MIC. As shown in Table 1, the basal levels of qnrS1 were similar in all of the plasmids harboring different lengths of the upstream sequence (pMG322, pMG323, and pMG327 to pMG330). Ciprofloxacin induction of qnrS1 occurred in plasmids pMG322 and pMG323, which contain 359- and 209-bp sequences, respectively, upstream of qnrS1. In contrast, no induction occurred when the qnrS1 upstream sequence was 172 bp or shorter (pMG327 to pMG330). These results indicate that a minimum of 209 bp of upstream sequence was required for induction.
We further analyzed the sequences upstream from the qnrS1 gene and constructed three plasmids, pMG324, pMG325, and pMG326, in which different parts of the area from −209 to −189 were deleted (Table 1). The transcript levels of qnrS1 from these plasmids were measured in E. coli DH10B (recA mutant). Ciprofloxacin treatment of bacteria carrying pMG324 produced 5-fold-increased levels of qnrS1 transcripts, while no induction was present for plasmids pMG325 and pMG326 (Table 1). These results indicate that in the essential 209-bp upstream region, sequence at position −204 to −189 is important for the ciprofloxacin induction of qnrS1.
Screen for candidate proteins binding to the upstream region of qnrS1.In order to determine the potential regulatory protein(s) that might control qnrS1 expression, we incubated cell extracts with bead-bound 359 bp of qnrS1 upstream DNA sequence. Proteins eluted from beads were separated on SDS-PAGE gels, and the protein bands were identified by mass spectrometry. Six candidate binding proteins, Dps, Fis, Ihf, Lrp, CysB, and YjhU, were detected. In addition, candidate binding sites for Ihf and DnaA (Fig. 1) (20) were found within the 359-bp upstream sequence. A putative DnaA binding sequence occurs at position −36 to −45 on the noncoding strand and abuts the proposed −35 promoter sequence on the coding strand, and two other potential DnaA binding sequences are located within the essential upstream region.
Expression of candidate regulatory proteins in response to ciprofloxacin.To determine whether the expression of these candidate qnrS regulatory proteins, identified either by binding from cell extracts or by putative binding sequences, were affected by ciprofloxacin exposure, we measured relative transcript levels by quantitative reverse transcription-PCR (qRT-PCR) in the presence and absence of sub-MICs of ciprofloxacin in strains J53 AziR and DH10B with pMG306. Little change in transcript levels (0.44- to 1.37-fold) of dps, fis, ihfA, ihfB, lrp, cysB, yjhU, and dnaA in either strain background was detected (Table S1).
Identification of chromosomal genes and plasmid sequences affecting ciprofloxacin induction of qnrS1 using a phenotypic screen.In order to determine proteins necessary for ciprofloxacin induction, we undertook an unbiased screen of pooled Keio collection mutants for reduced ciprofloxacin induction of pMG331, a reporter plasmid with 359 bp of sequence upstream from qnrS1 fused to lacZ. The screen identified three candidates that consistently deviated from wild-type controls and exhibited either an increase or decrease in β-galactosidase upon exposure to ciprofloxacin (Table 2). These three candidates were then determined by inverse PCR to be deletions in ihfA, ihfB, and cysB and were independently confirmed by testing the specific Keio collection knockout mutants introducing pMG331 and measuring both reporter transcript levels by qRT-PCR and β-galactosidase expression. ihfA and ihfB mutants exhibited decreased ciprofloxacin induction, while the cysB mutant exhibited increased induction. In similar experiments using the Keio collection dps, fis, lrp, and yjhU mutants, no effect on ciprofloxacin induction of the pMG331 LacZ reporter was found.
Relative expression levels of pMG331 reporter in E. coli parental strains and mutants
Role of DNA gyrase in SOS-independent ciprofloxacin induction of qnrS1.It was presumed that the SOS-independent ciprofloxacin induction of qnrS1 was the result of its interaction with DNA gyrase, as is the case with SOS-dependent induction. To establish the involvement of DNA gyrase in this induction, we also compared induction with plasmid pMG331 in strain J53 and its single-step gyrA (Ser83Leu) quinolone-resistant mutant and found that at the same ciprofloxacin concentrations, induction seen in J53 AziR was abolished in the mutant (Table 3). Thus, ciprofloxacin acts as an inducer through its proximal interaction with gyrase, and induction events proceed as a consequence of this interaction.
Effects of dnaA and gyrA on ciprofloxacin induction of expression of qnrS1 from reporter plasmid pMG331
Because dnaA mutants are not included in the Keio collection due to their lethality, we further investigated the role of DnaA in ciprofloxacin induction of qnrS expression using pMG331. Following introduction of plasmid-cloned dnaA or an empty vector plasmid into the strain containing the lacZ reporter, ciprofloxacin induction of lacZ was reduced by the dnaA plasmid, which was confirmed to have 50-fold increases in dnaA transcripts but was unaffected by the vector alone (Table 3). We also tested two dnaA temperature-sensitive strains, BM215 and CM742, for levels of reporter lacZ expression upon ciprofloxacin exposure at permissive and nonpermissive temperatures (Table 3). Across three sub-MICs of ciprofloxacin for both temperature-sensitive strains, levels of induction were slightly higher at nonpermissive temperature (42°C) than at permissive temperature (30°C). As a control for the adequacy of the temperature shift conditions, we tested in the absence of ciprofloxacin the expression of rpoH, a gene for which DnaA is known to behave as a repressor (21), and we found a 4-fold increase in expression at nonpermissive temperature (data not shown). Thus, dnaA acts on ciprofloxacin induction of qnrS in a manner consistent with that of a repressor.
In addition, during the Keio collection screen, eight mutants were identified as having reduced β-galactosidase induction by ciprofloxacin but for which independent introduction of the pMG331 reporter into the specific Keio collection mutant did not confirm loss of induction. To test for possible spontaneous changes in the reporter plasmid, we outcrossed those plasmids to wild-type strain BW25113 and found reduced induction to be plasmid associated. Sequencing upstream of qnrS1 identified deletions between −156 and −142 (TGCCAATGCAAGAG) in one mutant and −157 to −144 (GTGCCAATGCAAG) in two mutants, and the five remaining mutants had the shortest deletions from −156 to −144 (TGCCAATGCAAG).
Binding of the regulatory proteins to the DNA upstream of qnrS.To determine if IhfA, IhfB, CysB, and DnaA acted directly on the regulatory region upstream of qnrS, purified proteins were assessed for binding to the 359-bp upstream region with an electrophoretic mobility shift assay (EMSA). Using a biotinylated DNA fragment, each protein's binding capacity was tested at various concentrations, and a 1:1 mixture of IhfA and IhfB was also tested. We found that a mixture of IhfA and IhfB bound specifically to the 359-bp region upstream of QnrS (Fig. 2A), as did DnaA (Fig. 2B). No binding of CysB could be demonstrated.
DNA mobility shifts of qnrS1 upstream DNA for IhfAB and DnaA. (A) Interaction of the IhfAB protein with the qnrS promoter region. Lanes: 1, labeled qnrS promoter fragment (1 ng, 4 fmol); 2, labeled DNA with IhfAB (10 ng, 625 fmol) plus 200-fold excess unlabeled nonspecific DNA (salmon sperm DNA); 3, labeled DNA with IhfAB plus 200-fold excess of specific DNA (unlabeled qnrS promoter fragment); 4, labeled DNA with IhfAB. (B) Interaction of the DnaA protein with the qnrS promoter region. Lanes: 1, labeled qnrS promoter fragment (6 ng, 23 fmol); 2, labeled DNA with DnaA (3 μg, 58 pmol); 3, labeled DNA with DnaA plus 200-fold excess specific DNA (unlabeled qnrS promoter fragment); 4, labeled DNA with DnaA plus 200-fold excess nonspecific DNA (salmon sperm DNA).
DISCUSSION
Expression of qnrB, qnrD, and probably qnrE (based on sequence data) and qnr in Serratia marcescens (18) is triggered by ciprofloxacin and other DNA-damaging agents through the SOS system, which serves to protect bacteria from DNA damage effects and allows repair to occur (22). Quinolones inhibit DNA gyrase and DNA topoisomerase IV in bacterial cells and form enzyme-DNA complexes that block DNA replication and trigger the SOS response. Qnr proteins protect these target enzymes from inhibition of quinolones. LexA is a core regulator of the SOS system. qnrB, qnrD, and qnrE alleles have upstream LexA binding sites, and qnrB and qnrD expression is induced by quinolone exposure in a LexA- or RecA-dependent manner (18, 23).
The characteristics of qnrS1 expression, however, differ from those of qnrB, qnrD, and qnrE expression. qnrS1 is closely related to other qnr genes on the chromosomes of Vibrio species, such as V. parahaemolyticus, V. mytili, and V. ostreicida (96 to 97% amino acid identity), which may be reservoirs from which qnrS was originally mobilized to plasmids. Expression of qnrVS1 itself in V. splendidus is induced by ciprofloxacin but is not induced by mitomycin C or other DNA-damaging agents (16), and ciprofloxacin induction of qnrVS1 and plasmid-carried qnrS1 occurs in lexA mutant as well as recA mutant E. coli. In addition, there are no putative LexA binding sites found in the sequences upstream of both qnrS1 and qnrVS1. The regions upstream of qnrS1, however, differ substantially from the 200 bp upstream of qnrVS1 (53% identity with 33% gaps) (see Fig. S1 in the supplemental material). Notably, the 200-bp sequences upstream of qnr in V. parahaemolyticus, V. mytili, and V. ostreicida are much more closely related to the qnrS1 upstream sequence than that of V. splendidus (Fig. S2) and also lack LexA boxes and contain putative binding sites for DnaA and Ihf, suggesting differences as well as similarities in the regulatory components that underlie ciprofloxacin induction in E. coli and the various Vibrio species. Our findings focused on qnrS1 plasmid gene regulation in E. coli, since such plasmids are found in clinical isolates, and we confirm that ciprofloxacin induction of qnrS1 occurs independently of SOS. Thus, the mechanism of ciprofloxacin induction of qnrS1 and qnrVS1 is novel and might function to protect from some quinolone-like molecules that occur in nature (24). DNA damage from UV irradiation in a lexA SOS-defective mutant has been associated with small increases (2-fold) in expression of a number of genes, particularly those associated with the DNA replication machinery, nucleotide metabolism, heat shock, and chaperone proteins (25). The largest effects (5- to 20-fold) were found with nrdA and nrdB, which encode a ribonucleotide reductase controlled by DnaA (26). Non-LexA-dependent regulatory modules involved in response to DNA damage, including that by quinolones, have also included, among others, the global regulator Hns, the iron metabolism regulator Fur, and notably DnaA, as discussed below (27).
The elevation of the expression level of qnrS1 observed after ciprofloxacin exposure is not explained by a change in the qnr gene dosage or plasmid copy numbers (16). In order to explore the mechanisms involved in this SOS-independent induction of qnrS1, we constructed a series of recombinant plasmids, each containing the entire coding region of qnrS1 and different lengths of the upstream sequence, and measured induction levels of qnrS1 in a recA mutant E. coli strain (DH10B). The absence of induction in recombinant plasmid pMG327 and its presence in plasmid pMG324 indicated that a region within the upstream sequence from −204 to −172 is essential for ciprofloxacin induction. Within this region, deletion of sequences at positions −200 to −189 (CCTAACCCTATC) in plasmids pMG325 and pMG326 also eliminated induction. Notably in the Keio collection screen for loss of induction phenotypes, we unexpectedly found several independent spontaneous deletions in a nearby upstream region of −156 to −144 (TGCCAATGCAAG), further strengthening the data for the importance of these regions for ciprofloxacin induction of qnrS1 expression.
To investigate potential regulatory elements involved in ciprofloxacin induction of plasmid-carried qnrS1, we used 359 bp of upstream DNA sequence to identify binding proteins in cell extracts. Six proteins were identified by mass spectrometry to bind this DNA region: Dps, Fis, Ihf, Lrp, CysB, and YjhU. Knockout mutants of these proteins from the Keio collection of E. coli mutants were then tested for induction, but only deletions in ihfA, ihfB, and cysB exhibited a change in induction pattern after ciprofloxacin exposure. The role of the other proteins that appeared to bind to DNA upstream of qnrS in regulation of qnrS expression is uncertain and might represent nonspecific binding or an ancillary but nonessential role in qnrS1 expression. We also screened other Keio collection mutants for their effects on ciprofloxacin induction of qnrS1 based on their previously reported changes in expression after exposure to ciprofloxacin, including mutants in recN, rssB, ruvB, uvrA, and uvrD (28). None, however, affected ciprofloxacin induction of qnrS1 (data not shown).
The Keio collection screen also independently identified a dependence of ciprofloxacin induction on intact IhfA and IhfB and a hyperinduction phenotype in cysB mutants. The independent identification of these three genes further supports their role in the mechanism of ciprofloxacin induction. No other novel genes were identified in this screen, possibly due to the inability to test essential genes and potential redundancies in the induction mechanism that cannot be detected in single-gene deletion library screens. The effects of DnaA would not have been detected from our screen because of the essential nature of this protein that eliminates its presence in the Keio collection, but based on the finding of putative DnaA binding sites in the 359-bp upstream region, we tested direct binding of DnaA to this fragment and the effects of plasmid overexpression of dnaA and temperature inactivation of DnaA in two temperature-sensitive dnaA mutants on reporter gene expression and found effects consistent with DnaA acting directly as a repressor of ciprofloxacin induction. DnaA has a known role in non-SOS-dependent regulation of gene expression in response to DNA damage, which occurs with ciprofloxacin treatment, in addition to its known role in initiation of DNA replication (27, 29). Notably, dnaA expression is induced by ciprofloxacin in a LexA-independent manner (29).
IhfA and IhfB function as a dimer, and IhfAB bound specifically to the 359-bp sequence upstream of qnrS1 that contains candidate binding sites. IhfAB is a global regulatory protein that helps maintain DNA architecture and affects processes such as DNA replication, recombination, and the expression of many genes. Notably, Hns, which is involved in SOS-independent gene expression responses to DNA damage (27), also has a broad role in maintaining DNA architecture and modulating DNA complex formation with other proteins (30). The mechanism by which IhfAB enables ciprofloxacin induction without affecting baseline expression of qnrS1 is uncertain. The roles of Ihf in bacterial physiology are broad, with Ihf having been shown to bind at many targets, with its major function being to bend DNA sharply to allow transcription in all phases of growth (31). Ihf has also been shown to enable binding of DnaA to weak DnaA binding sites in initiation of DNA replication (32). Although both DnaA and IhfAB bind directly to DNA upstream of qnrS, it is not clear that they act in concert in a similar manner, since the presence of DnaA reduces induction and the presence of IhfAB increases it. Thus, IhfAB might in this case function to reduce rather than enhance binding of DnaA.
CysB is a tetrameric helix-turn-helix-type dual transcriptional regulator. Although it has a function in the biosynthesis of cysteine, it also controls the transcription of an operon involved in novobiocin resistance and transcription of genes involved in sulfur utilization and sulfonate-sulfur catabolism via cysteine biosynthesis. No apparent CysB binding motifs (33) were found in the 359-bp sequence upstream of qnrS1, suggesting that it acts indirectly. The mechanism of its effects on qnrS1 induction, however, remains unclear.
Both Ihf and CysB are known to be involved in the pH response in E. coli (34). The relative qnrS reporter gene transcription levels, however, did not change in either wild-type or mutant strains when exposed to shifts to acidic (pH 5.5) or basic (pH 8.5) conditions (data not shown). Similarly, other Keio collection mutants of genes involved with pH response (including adiA, cadA, cadC, and nhaA) showed no relative change in qnrS1-lacZ reporter gene expression when exposed to ciprofloxacin (data not shown).
In summary, the expression of plasmid-carried qnrS1 is induced by ciprofloxacin in a gyrase-dependent and SOS-independent manner that requires intact IhfA and IhfB and is affected by CysB and DnaA. IhfAB and DnaA appear to act directly and CysB indirectly. This induction is also dependent on select upstream elements. The mechanism as yet does not appear to involve binding of drug to a specific repressor, as occurs with tetracycline binding to a TetR repressor that results in increased expression of tet resistance determinants (35), and additional studies are needed to define the role of CysB and the details of the induction mechanism. Thus, resistance conferred by both SOS-dependent qnr genes and SOS-independent qnrS is enhanced by ciprofloxacin exposure, but the natural functions and natural inducers of qnr genes remain elusive.
MATERIALS AND METHODS
Bacterial strains and growth conditions.Bacterial strains in this study are summarized in Table 4. All E. coli strains were grown at 37°C in Mueller-Hinton broth (MHB; Difco) or on Mueller-Hinton agar (MHA; Difco). The Keio collection was used for the loss-of-induction screen. This collection comprises 3,985 nonessential, in-frame, single-gene deletions of E. coli K-12 strain BW25113 (36). These deletion strains, which retain a kanamycin resistance marker, were grown at 37°C in Luria-Bertani (LB; Difco) agar or MHB with 30 μg/ml kanamycin for all experiments.
Bacterial strains and plasmids used in this study
Chemicals.Ampicillin, chloramphenicol, ciprofloxacin, and kanamycin were obtained from Sigma-Aldrich (St. Louis, MO).
Antimicrobial susceptibility testing.Ciprofloxacin MICs were determined on MHA by Etest (bioMérieux, Durham, NC).
Identification of putative binding sites.Proposed promoter sequences were identified using BPROM (www.softberry.com), and DnaA, Ihf, CysB, and other potential binding sites were located using Virtual Footprint with the PRODORIC library (prodoric.de) (20).
Transformation of plasmid with qnrS1 into E. coli recA mutant.A clinical plasmid, pMG306, carrying the qnrS1 gene and an insertion of a selectable chloramphenicol acetyltransferase gene (37) was extracted from E. coli J53 AziR pMG306 by a QIAprep spin miniprep kit (Qiagen Inc., Valencia, CA). The pMG306 plasmid was transformed by electroporation into E. coli DH10B, a recA mutant defective in the SOS response, selecting transformants on LB agar containing 20 μg/ml chloramphenicol.
Construction of truncated qnrS1 plasmids with various upstream sequences.The intact qnrS1 gene and 359 bp of upstream sequence from the start codon were amplified from pMG306 and cloned into pUC18 in an orientation opposite that of the lac promoter of pUC18 in order to avoid its influence on transcriptional activity of the native promoter. The plasmid generated was named pMG322. In a similar manner, three additional plasmids were constructed to contain the qnrS1 gene and 209 (pMG323), 172 (pMG327), 126 (pMG328), 92 (pMG329), and 43 (pMG330) bp of upstream sequence from the start codon of qnrS1. The recombinant plasmids were then introduced into E. coli DH10B by electroporation, selecting transformants on LB agar containing 100 μg/ml ampicillin.
Site-directed mutagenesis of qnrS1 plasmids.Three site-directed mutants of plasmid pMG322 were generated using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA) according to the manufacturer's instructions. The primers were chosen for introducing 5- or 6-bp deletions at three sites and generated deletions at upstream sites −209 to −205 (pMG324), −200 to −194 (pMG325), and −194 to −189 (pMG326) (Fig. 1).
Exposure of DH10B strains to ciprofloxacin.E. coli strains carrying a qnrS1 plasmid were grown overnight at 37°C in MHB with 100 μg/ml ampicillin and suspended in fresh MHB to achieve a bacterial density of 1 × 108 CFU/ml. Each bacterial suspension was then diluted 100-fold and incubated with shaking at 37°C until the cultures reached exponential growth at an optical density at 600 nm (OD600) of 0.1 to 0.2. Ciprofloxacin was added to the aliquots of the culture to a final concentration of one-half the respective MICs. The bacterial cells were incubated for 30 min at room temperature without shaking. After centrifugation at 3,500 × g for 5 min at 4°C, the pellet was subjected to RNA extraction.
RNA extraction and quantitative real-time PCR of qnrS1.Bacterial mRNA was extracted with the RNeasy minikit (Qiagen, Valencia, CA), followed by digestion with a Turbo DNase-free kit (Ambion, Austin, TX) to remove contaminating DNA, and then was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). cDNA was synthesized using 100 ng of total mRNA with a Verso cDNA kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. qRT-PCR for qnrS1 was carried out with the SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA) and the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA), with cycles of 20 s at 95°C, followed by 40 cycles of 3 s at 95°C and 3 s at 60°C. The qRT-PCR for lacZ was carried out after exposure to ciprofloxacin with a method identical to that for the β-galactosidase assays and then using cycles of 15 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 52.5°C. All primers used in this study were designed using Primer3 (version 0.4.0) software (http://frodo.wi.mit.edu/primer3/) and synthesized at the Massachusetts General Hospital DNA Core Facility (Cambridge, MA) or Eton Bioscience (Charlestown, MA) (see Table S2 in the supplemental material). Relative expression levels were calculated using the ΔΔCT method with expression of mdh used as an internal control, as expression of mdh did not change in response to ciprofloxacin (5). Each experiment was performed at least in triplicate, and at least two independent culture replicates were performed.
Identification of bacterial proteins binding to the qnrS1 upstream region.The identification of specific DNA sequence binding proteins was performed as previously described, with modifications (38). A 359-bp DNA fragment of sequence upstream of qnrS1 (Fig. 1) was amplified by PCR using a purified biotinylated primer (IDT, Coralville, IA), separated by agarose gel, and purified by Freeze ‘N Squeeze DNA gel extraction spin columns (Bio-Rad, Hercules, CA). Twenty μg DNA was immobilized on 2 mg of magnetic beads with covalently coupled streptavidin (Dynabeads M-280; Dynal) according to the manufacturer's instructions. DNA-linked beads were incubated with 200 μg of protein extract in 800 μl of binding buffer (10 mM HEPES, pH 8.0, 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 0.25 mM dithiothreitol) containing 1 μg of poly(dI · dC), 600 ng of sheared herring sperm DNA, and 10% glycerol for 15 min at room temperature. Beads were washed once with binding buffer containing 5 ng/μl of herring sperm DNA and twice with binding buffer. Proteins were eluted in 100 μl of binding buffer containing 0.5 M NaCl and dialyzed against water for 1 h. The proteins were separated on an SDS-PAGE gel (12%), and protein bands were cut from the gel and identified by mass spectrometry (Taplin Mass Spectrometry Facility, Harvard Medical School, Boston, MA).
Plasmid construction of qnrS1 upstream region fusion to lacZYA operon in pRS415 vector.The 359-bp upstream sequence of qnrS1 (GenBank accession number JX102659) was amplified from pMG306 and cloned into pRS415 in frame to the lac promoter (39). The plasmid generated, named pMG331, was transformed by electroporation into E. coli DH5α, and transformants were selected on LB agar containing 100 μg/ml ampicillin. The plasmid was then extracted, confirmed through sequencing, and transformed by electroporation into E. coli BW25113, the background strain used in the Keio Collection. Transformants were selected on LB agar containing 100 μg/ml ampicillin.
Keio Collection batch transformation with pMG331 for screening.Samples (5 μl) from microtiter plate wells containing individual mutants of the Keio Collection were pooled (48 wells/pool) in 20 ml of LB with 30 μg/ml kanamycin and incubated overnight at 37°C. A 1:10 dilution of the overnight cultures in LB with 30 μg/ml kanamycin was grown to exponential phase, centrifuged at 4,000 rpm, washed four times with cold H2O, and resuspended in 10% glycerol. Aliquots of 100 μl of each pool were then electroporated with pMG331, and transformants were selected on LB agar with 100 μg/ml ampicillin and 30 μg/ml kanamycin. At least 128 colonies were then patched onto LacTZ agar plates (0.01% tryptone, 0.001% yeast extract, 0.015% Bacto agar, 85 mM NaCl, 1% lactose, 0.005% 2,3,5 triphenyl tetrazolium chloride) with 100 μg/ml ampicillin, 30 μg/ml kanamycin, and 0.002 μg/ml ciprofloxacin together with three controls: parental BW25113 electroporated with pRS415, pMG331, or, as a noninducible negative control, pMG332 (a derivative of pMG331 that is missing region −200 to −188 [CCTAACCCTATC], which is critical for ciprofloxacin induction of qnrS). Of these independent patch colonies, those that resembled the pinkish-red phenotype of pRS415 missing the 12-bp critical upstream region were isolated for screening in a β-galactosidase assay.
Keio Collection β-galactosidase assay phenotype screen.The β-galactosidase assay was performed by growing duplicate 1-ml overnight cultures of prescreened candidate strains alongside the three control strains described in the previous section in a 2.2-ml polypropylene 96-deep-well plate (Thermo Scientific, Wilmington, DE) with 100 μg/ml ampicillin and 30 μg/ml kanamycin by following a modified protocol (40). The strains were then grown to an OD600 of 0.4 to 0.5 after a 100-fold dilution into a fresh 96-deep-well plate and incubated with ¼ MIC of ciprofloxacin for 30 min at room temperature. Growth was arrested with 30 μg/ml chloramphenicol, and the density of the cultures was measured using a 96-well flat-bottom plate (USA Scientific, Ocala, FL) and read on a VMax kinetic Maxline microplate reader (Molecular Devices, Sunnyvale, CA) with an OD600 filter. The cells were then lysed using PopCulture reagent (EMD Millipore), and a 10-fold dilution of the lysed cells was performed in buffer A (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 40 mM β-mercaptoethanol). Using independent culture and internal replicates in 96-well microtiter plates, the β-galactosidase reaction was initiated with 2.5 M 2-nitrophenyl β-d-galactopyranoside (ONPG) and stopped with 300 mM Na2CO3 at a time point that provided sufficient ONPG cleavage to read in a spectrophotometer (at 420 μm), and reaction times were recorded. β-Galactosidase activity (in Miller units) was then calculated using a standard equation (41).
Inverse PCR of candidate mutants with reduced ciprofloxacin induction of β-galactosidase.In order to determine the transposon insertion site of screened mutants identified as having altered ciprofloxacin induction, genomic DNA was extracted from the candidate Keio collection strains identified in the β-galactosidase assays using an Easy-DNA gDNA purification kit (Invitrogen, Carlsbad, CA) and suspended in water. To produce microplasmids, 1.5 μg of genomic DNA was then digested in a 10-μl reaction volume using either AgeI, BspHI, or MluI (New England BioLabs, Ipswich, MA), restriction enzymes that produced sticky ends, did not digest the kanamycin resistance determinant and were predicted to produce 1-kb average-sized fragments of the E. coli K-12 genome. The digestion was carried out for 4 h and inactivated for 20 min at the enzyme-specific temperatures. The 10-μl digestion was then ligated in a 100-μl volume at 25°C for 16 h using T4 DNA ligase (New England BioLabs, Ipswich, MA) and cleaned with a QIAquick PCR purification kit (Qiagen, Valencia, CA) in 50 μl of TE buffer. Different sets of primers within the resistance determinant that amplify outward toward the adjacent genes were synthesized (Table 2; Table S2). Inverse PCR was performed using Maxima Hot Start Taq DNA polymerase (Thermo Scientific, Wilmington, DE), a 25-fold dilution of the ligation product, and a final primer concentration of 0.75 μM on a C1000 thermal cycler (Bio-Rad, Hercules, CA) using a 94°C denaturing step for 2 min followed by 30 cycles of 94°C for 30 s, annealing for 35 s, and extending for 100 s at 72°C. Amplicons were detected through gel electrophoresis in a 1% agarose gel and submitted for sequencing to Tufts University Core Facility (Boston, MA).
Candidate gene cloning.ihfA, ihfB, cysB, and dnaA were amplified by PCR and cloned into expression vectors, either pTrcHisC with transformation into E. coli DH5α or pET28a(+) with transformation into E. coli BL21(DE3). Vectors and amplicons were doubly digested with appropriate restriction enzymes and ligated with T4 ligase. Orientation, reading frame, and integrity of cloned genes were confirmed through plasmid sequencing.
Expression and purification of histidine-tagged proteins.E. coli cells with the cloned genes of interest were grown in MHB at 37°C (OD600 of 0.8) and then exposed to 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h. The cells were then centrifuged at 10,000 × g for 45 min at 4°C and resuspended in buffer TN (50 mM Tris, pH 7.5, 500 mM NaCl) at a 0.05 volume of the original culture and sonicated on ice for six 30-s cycles with 30-s intermittent rest periods. Benzonase nuclease (Sigma-Aldrich) and 1× ProteoGuard complete protease inhibitor cocktail (Clontech, Mountain View, CA) were added to the lysate and incubated on ice for 30 min, followed by centrifugation at 10,000 × g for 45 min at 4°C and filtering the supernatant through a Steriflip-GP, 0.22-μm, polyethersulfone membrane (EMD Millipore, Billerica, MA). Purification of the histidine-tagged proteins was performed by nickel affinity chromatography (GE Healthcare, Marlborough, MA), using 1-ml fractions and incremental concentrations of imidazole. The fractions were run on SDS-PAGE gels for Coomassie staining and Western blotting (Invitrogen, Carlsbad, CA). The appropriate fractions were then cleared of imidazole using PD-10 desalting columns (GE Healthcare, Marlborough, MA) and concentrated on Amicon Ultra-15 centrifugal filter units (EMD Millipore).
Purification of DnaA followed the method of Smith and Grossman (42), with some modifications. The His6-DnaA was overexpressed in E. coli BL21 at 30°C and an OD600 of ∼0.4, and then 0.4 mM IPTG was added and incubated for 3 h. The 500-ml sample was centrifuged at 8,000 rpm for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended with 10 ml of Talon equilibration buffer with 5 mM imidazole and stored at −80°C. The sample was thawed at room temperature, and one tablet of protease inhibitor cocktail (cOmplete ultra tablets; Sigma-Aldrich, St. Louis, MO), MgCl2 to a final concentration of 10 mM, and 2 μl of Benzonase were added. The mixture was stirred for ∼20 min until the viscosity decreased and centrifuged at 10,000 rpm for 20 min at 4°C. The lysed supernatant was filtered and applied to the preequilibrated 5-ml Histalon (TaKaRa Bio, Mountain View, CA) column. The column was washed with 5 column volumes of equilibration buffer, washed with 10 mM imidazole wash buffer, and eluted with 150 mM imidazole elution buffer. The purified sample was desalted with a PD-10 column (GE Healthcare) and concentrated with Amicon Ultra 10K (EMD Millipore). The concentration was measured using a NanoDrop spectrophotometer (ND1000 V3.6.0).
Electrophoretic DNA mobility shift assays.Using a LightShift chemiluminescent EMSA kit (Thermo Scientific, Wilmington, DE), the binding of CysB, DnaA, and a combination of IhfA and IhfB to the labeled 359-bp region upstream of qnrS1 (GenBank accession number JX102659) was assessed. This fragment was amplified using a biotinylated 5′ primer (Integrated DNA Technologies, Coralville, IA) and diluted to ∼1 fmol per reaction. LightShift chemiluminescent EMSA kit controls were run alongside mixtures of biotinylated DNA and various protein concentrations (0.1 μg to 1 μg) on an acrylamide nondenaturing gel, transferred to a 0.45-μm nylon membrane (Thermo Scientific, Wilmington, DE), and detected on autoradiography film (GE Healthcare, Marlborough, MA). Specificity of binding was determined by addition of 100- to 200-fold excess of specific unlabeled DNA in comparison to the same excess of nonspecific salmon sperm DNA. DnaA mobility shift studies were performed similarly except for preincubation of DnaA protein with ATP, as previously described by Bonilla and Grossman (43).
Accession number(s).The intact coding region of qnrS1 and 359-bp upstream sequence was deposited in GenBank under accession number JX102659. Comparison sequences include V. splendidus WP_061032006 and nucleotides 126738 to 127097 in NZ_LNRO01000003, V. parahaemolyticus WP_029823919 and nucleotides 20247 to 20606 in NZ_MSEH01000005, V. mytili WP_041155100.1 and the reverse complement of nucleotides 864 to 1223 in NZ_JXOK01000026.1, and V. ostreicida WP_076586686 and nucleotides 416386 to 415745 in NZ_MPHM01000002.
ACKNOWLEDGMENTS
This work was supported in part by a grant to Y.F. from the National Natural Science Foundation (31370164), China, and by grant R01 AI057576 (to D.C.H. and G.A.J.) from the National Institutes of Health, U.S. Public Health Service.
FOOTNOTES
- Received 21 April 2018.
- Returned for modification 22 May 2018.
- Accepted 5 June 2018.
- Accepted manuscript posted online 18 June 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00806-18.
REFERENCES
- Copyright © 2018 American Society for Microbiology.