1 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322; 2 Laboratories of Microbial Pathogenesis, VA Medical Center, Decatur, GA 30033, USA
Received 10 January 2002; returned 4 July 2002; revised 5 August 2002; accepted 9 October 2002
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Abstract |
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Introduction |
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Expression of the mtrCDE operon is controlled by a transcriptional repressor, MtrR, the product of the divergently transcribed mtrR gene.2,3,12 It has been shown that MtrR binds within the 250 bp intergenic region, between mtrR and mtrC that accommodates the promoter for mtrCDE transcription, thereby downregulating expression of the efflux pump operon.3,12,13 Mutations in mtrR, or in the promoter region of the gene, result in increased resistance of gonococci to antimicrobial hydrophobic agents (HAs) that are substrates of the pump.2,3,12,14 Conversely, mutations in mtrCDE result in hypersusceptibility to HAs,3,5,6,15 and can phenotypically suppress mutations in mtrR that would normally increase resistance to HAs. Originally, strains bearing mutations that caused HA hypersusceptibility were termed envelope mutants.16 In one study,17 they represented at least 15% of all clinical isolates.
We have studied HA-resistant and HA-hypersusceptible clinical isolates, and laboratory-derived mutants, in order to identify genes that determine the levels of gonococcal susceptibility to antimicrobials that can penetrate to mucosal surfaces infected by gonococci. Through such analyses, we have identified several mutations in mtrR that would change repressor activity,14 or mtrR transcription,14,1820 generating an HA-resistant phenotype. During a recent study of HA-hypersusceptible clinical isolates, we noted a particular strain that did not harbour a mutation(s) in mtrCDE, but did contain a phenotypically suppressed mtrR mutation. The suppressor mutation was located to a novel gene positioned downstream of mtrR that encodes a putative cytoplasmic membrane protein (CMP). Because this gene is closely linked to mtrR, is subject to MtrR repression, and is needed for high-level resistance to certain HAs, we designated it mtrF. Orthologues of mtrF were detected in several Gram-negative and -positive bacteria, suggesting that the predicted products may represent a heretofore undescribed protein family involved in antimicrobial resistance.
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Materials and methods |
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The gonococcal strains used in this study are described in Tables 1 and 3. HA hypersusceptibility of gonococci is defined as an MIC of 0.12 mg/L of erythromycin and
62 mg/L of Triton X-100 (TX-100).15 Strains were routinely grown as non-piliated, opacity-negative colony variants on GCB agar (Difco, Detroit, MI, USA), containing glucose and iron supplements21 at 37°C under 3.8% (v/v) CO2.
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Chromosomal DNA extractions were performed as described by McAllister & Stephens.22 PCR amplifications, with chromosomal DNA as template, were performed as described previously.2 DNA sequencing of PCR products was performed using the cycle sequencing protocol,2 or by automated sequencing using the Nucleic Acid Sequencing Core Facility of Emory University. DNAStar was used for nucleotide and amino acid sequence analysis and alignments. Protein topology predictions were generated using the algorithm of von Heijne,23 as implemented by the TopPred II program,24 and were confirmed by several other web-based topology prediction programs. Putative proteins, demonstrating pronounced similarity to MtrF, were identified through searches of the National Center for Biotechnology Information (Bethesda, MD, USA) finished and unfinished genomes (http://www. ncbi.nlm.nih.gov/BLAST) using the Basic Local Alignment Search Tool (BLAST) program TBLASTN.25 Preliminary sequence data used in similarity comparisons were obtained from The Institute for Genomic Research website at http://www.tigr.org. Gonococcal sequencing data were obtained from the Gonococcal Genome Sequencing Project website at http://www. genome.ou.edu. Protein motifs were defined with the aid of the MEME26 and MAST27 programs.
Southern blotting
DNA probes were generated by PCR amplification and labelled using the Roche (Indianapolis, IN, USA) Non-radioactive DNA Genius 2 Labeling Kit, according to the manufacturers directions. Chromosomal DNA (3 µg) was digested with ClaI. DNA fragments were separated by agarose gel electrophoresis and blotted on to nylon filters; hybridization, washes and detection were performed, as described in the Genius System Users Guide for Membrane Hybridization, using anti-digoxigenin alkaline phosphatase conjugate, and CSPD (Roche).
Transformation experiments
Piliated gonococci were transformed with chromosomal DNA, or agarose gel electrophoresis-purified PCR products, essentially as described by Gunn & Stein.28 Kanamycin-resistant (KmR) transformants were selected using kanamycin 50 mg/L (Sigma Chemical Company, St Louis, MO, USA). Hypersusceptible mutants were identified by preliminary screening for lack of growth on plates containing TX-100 at 500 mg/L. Antibiotic MICs for all strains were determined, as described previously;16 the MIC was defined as the point at which there was no longer confluent growth of the suspension spot.
Inactivation of mtrF
The 5' portion of the mtrF gene was amplified using primers that incorporated an EcoRI site at the 3' end. The 3' portion of the mtrF gene was amplified using primers that incorporated a HindIII site at the 5' end. The non-polar aphA-3 cassette29 was inserted into the engineered sites, following PCR amplification from pUC18K. The resulting construct was transformed into gonococcal strain FA19, as indicated above. Inactivation of mtrF was confirmed by PCR analysis, and a representative transformant (WV9) was used as a source of DNA for all subsequent transformation experiments involving inactivation of mtrF.
Analysis of mtrF gene expression
Using total RNA prepared from gonococci by the method described by Biran et al.,30 gene expression was quantified by reverse transcriptasePCR (RTPCR),31 using Superscript II reverse transcriptase (Gibco BRL, Carlsbad, CA, USA).
Cloning and expression of mtrF in Escherichia coli
The mtrF gene was inserted into the pBAD-TOPO expression vector (Invitrogen, Carlsbad, CA, USA), according to the manufacturers directions. PCR primers were designed such that the MtrF protein produced was in frame with the N-terminal leader peptide, and the mtrF stop codon was removed so as to fuse mtrF to the V5 epitope. Primers used were MTRFTOP1 (5'-ATGAGTCAAACCGACGCGCGT-3') and MTRFTOP2 (5'-AGGTGCGGGATACAAAGTGGGC-3'). In-frame cloning of MtrF was verified by sequencing of plasmids extracted from transformants of TOP-10 One-Shot competent cells (Invitrogen). Plasmid extractions were performed using the Qiagen Spin Miniprep Kit (Qiagen, Valencia, CA, USA), according to the manufacturers directions. E. coli abgT::Km and abgT+ strains (obtained from Dr Brian Nichols, University of Illinois at Chicago, IL, USA) were rendered competent using the CaCl2 procedure,31 and were transformed with the pBAD-MtrF construct, and selected on LB agar containing ampicillin 50 mg/L. A final arabinose concentration of 0.2% was used to induce MtrF production; cells were grown for 30 min, followed by 2 h of arabinose induction, before plating on M9 minimal medium in agarose containing different concentrations of p-aminobenzoyl-glutamate and/or harvesting for membrane extraction. Total cell envelope proteins were prepared as described by Clark et al.32 Proteins were separated by SDSPAGE33 and subsequently either stained with Coomassie Brilliant Blue or subjected to western blotting31 using the anti-V5 antibody (Invitrogen) for detection of the TOPO-pBAD-MtrF product.
Data deposition
The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF176820 and AF176821).
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Results |
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We15 demonstrated previously that the HA-hypersusceptible laboratory strains BR87 and BR54, of the FA19 genetic lineage,16 contain small (610 bp) deletion mutations in the efflux pump structural genes mtrC and mtrD. We also showed that these mutations phenotypically suppress mutations in the mtrR gene that normally confer increased resistance to antimicrobial HAs on gonococci. In order to determine whether mtrC and/or mtrD gene mutations are responsible for the antibiotic-hypersusceptible phenotype of clinical isolates, we examined strain EU75, a recent clinical isolate that is hypersusceptible to TX-100 (Table 1). The mtrR and mtrCDE loci were sequenced and the only mutation found was a base substitution in mtrR that would cause a missense mutation (Thr-39Ala-39) in the first helical domain of the helixturnhelix region in MtrR. Phenotypic suppression of the mtrR missense mutation in strain EU75 was verified by demonstrating that its chromosomal DNA, and an mtrR-specific PCR product obtained from this DNA, transformed wild-type strain FA19, yielding derivatives with increased resistance to HAs, such as Ery and TX-100 (see strain WV28 in Table 1). Moreover, DNA sequencing studies confirmed that transformant strain WV28 had acquired the mtrR allele from donor strain EU75 (data not presented).
To understand the genetic basis of phenotypic suppression of the mtrR mutation in N. gonorrhoeae EU75, we constructed HA-resistant (HAR) transformants of EU75, using chromosomal DNA from HAR strain FA140 (as FA19 but mtrR-140, penA, penB). Table 1 shows the resistance levels of a representative transformant, WV5, in comparison with recipient strain EU75 and donor strain FA140. We noted that the HA-resistance character of transformant strain WV5 more closely resembled those of gonococcal strains, having a single bp deletion in the mtrR promoter, rather than the intermediate HA-resistance phenotype, typically due to missense or nonsense mutations in the mtrR-coding sequence.3,13,14 As strain EU75 has a wild-type mtrR promoter sequence (data not presented), while strain FA140 has the promoter bp deletion, as well as a missense mutation at codon 45 (Gly-45 to Asp-45) in mtrR,15 we determined whether transformant strain WV5 had acquired the mtrR gene from donor strain FA140. DNA sequencing of the mtrR gene from strain WV5 revealed that it had indeed acquired the FA140 mtrR sequence (data not presented), indicating that a recombination event had occurred at the mtr locus in transformant strain WV5. Accordingly, we investigated whether there was evidence for gross genomic alterations in and around the mtr locus that might provide some indication of the genetic basis of HA hypersusceptibility in strain EU75. Results of Southern blotting analyses of ClaI-digested chromosomal DNA from gonococcal strains EU75, WV5, FA140 and FA19, using a labelled probe target to an open reading frame (degR) located immediately downstream from mtrR (Figure 1a), provided further evidence that a recombination event in or around the mtr locus had occurred in strain WV5. Thus, the single DNA fragment from EU75 detected by the probe was 500 bp larger than the DNA fragments of the other strains, including transformant WV5 (3.8 kb versus 3.3 kb) (data not presented).
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Given its close proximity to mtrR and the mtrCDE operon, we investigated whether orfX might be subject to MtrR transcriptional control, as is mtrCDE.12 To test this possibility, we performed RTPCR analysis of orfX and mtrD mRNA, using RNA extracted from isogenic strains FA19 and KH15. Strain KH15 contains the single bp deletion within the mtrR promoter that severely represses mtrR expression, while enhancing mtrD expression, but not expression of an unconnected gene, rmp.6 We found that expression of both mtrD and orfX, but not rmp, was elevated in strain KH15, suggesting that both are subject to MtrR control (Figure 3). We renamed orfX as mtrF, due to the close proximity of orfX to the mtrR and mtrCDE genes and its apparent regulation by MtrR.
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A number of unfinished and complete genome sequence databases were searched with the predicted MtrF protein sequence, using the TBLASTN algorithm,24 to identify any similar proteins. Proteins related to MtrF that showed significant similarity over the length of the entire protein, were found in several bacteria, nine of which are shown in Figure 4. Except for the AbgT protein of E. coli, all of the MtrF-like proteins are hypothetical. Their respective ORFs are found in both Gram-positive and -negative bacteria; percentages of identity range from 36% to a high of 97% for a putative protein from Neisseria meningitidis (Table 2). Figure 5 shows a phylogenetic tree of the full-length MtrF, and similar proteins, based upon the protein alignments. Despite repeated searches, no known functional domains or motifs have been found within MtrF. We, therefore, propose that MtrF and related proteins represent a new protein family.
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Consequences of mutations in mtrF
To determine the contribution of mtrF to HA resistance, we constructed a non-polar insertional mutation (mtrF::Km) in the mtrF gene of a number of gonococcal strains. Insertional inactivation of mtrF in wild-type strain FA19 (resulting in strain WV9) did not alter its level of HA susceptibility (Table 1). In contrast, inactivation of mtrF in the HAR strain FA140 (generating strain WV16) reduced its level of HA resistance, particularly to the non-ionic detergent TX-100. In contrast, susceptibility to nalidixic acid, which is not a substrate of the mtr pump, did not change, indicating that the decreased resistance observed in the mtrF mutant was not a general defect in antibiotic resistance.
To define further the role of mtrF in mtr-mediated HAR, genetic epistatic tests were conducted, by introducing the mtrF::Km mutation into a strain (BR54) containing a defined mutation in mtrD (mtrD-54); the reciprocal construction was generated by introducing the mtrD::Km mutation from strain KH14 into strain EU75 containing the mtrF-75 mutation (Figure 2). Since mutations in mtrF had been found not to decrease MIC levels to the same extent as mtrCDE mutations [compare BR54 (Table 3) with WV16 in Table 1], a comparison of the MICs for the resulting strains, WV12 (BR54 mtrF::Km) and WV14 (EU75 mtrD::Km), with those of the parental strains, was undertaken. The introduction of mtrF::Km into BR54 (mtrD-54) to produce strain WV12 did not alter its HA susceptibility (Table 3). However, when mtrD::Km was transferred into strain EU75 (mtrF-75) to generate strain WV14, two- to eight-fold reductions in the MICs of mtr substrates were observed (Table 3). These data indicate that MtrF and MtrCDE do not act independently to confer HAR; if this were the case, a decrease in the MICs of HAs for both strains, WV12 and WV14, compared with those of the parental strains, would be expected.
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Discussion |
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The discovery of a family of MtrF-like proteins, among both Gram-positive and Gram-negative bacteria, is striking. The MtrF-like proteins appear to fall into two clusters, which differ in extent of identity to the MtrF protein of gonococci. Cluster I consists of proteins from bacteria with MtrF-like proteins, having high (80100%) identity to MtrF, while cluster II accommodates those with much lower percentage identities (3648%). Except for Vibrio cholerae, which is predicted to produce an MtrF-like protein intermediate to these two clusters, the majority of MtrF-like proteins fall into cluster II. The AbgT protein of E. coli is the only one in the cluster with a proposed function,34 namely a transporter of p-aminobenzoyl glutamate.34 Although AbgT is one of the proteins more distantly related to MtrF (Figure 5), it was deemed to be important to determine whether MtrF functions in a manner similar to that of AbgT. To test for functional similarity, mtrF from strain FA19 was cloned into the pBAD expression vector. The resulting construct was introduced into an E. coli strain with an insertionally inactivated abgT gene. Even though we could demonstrate production of MtrF on induction, and that the expressed protein was localized to the membrane, the growth defect imparted by the abgT::km mutation, when cells were grown on low concentrations of p-aminobenzoyl-glutamate, was not relieved (data not presented). We conclude, therefore, that MtrF is not functionally equivalent to AbgT and cannot compensate for the loss of the latter in the E. coli strain. This probably reflects the lower identify between MtrF and AbgT, as compared with MtrF-like proteins in cluster I.
The high degree of amino acid identity, and existence of highly conserved motifs (Figure 4) among MtrF-like proteins, suggests that they define a previously undiscovered protein family. The evolutionary relationship of these MtrF-like proteins clearly deserves more study. However, it is noteworthy that almost all of the bacteria shown in Figures 4 and 5 that possess MtrF-like proteins, also encode variants of the gonococcal RND transporter MtrD (Table 2). Although the amino acid identities of some of these proteins with MtrD are fairly low, especially among Gram-positive bacteria, GenBank searches with one such MtrD protein from Staphylococcus aureus, revealed homology with many other RND transporters. These observations suggest the possibility that MtrF-like proteins in other bacteria might function in efflux-mediated antimicrobial resistance, in a manner similar to that discussed for gonococci. Accordingly, understanding the function of MtrF in gonococcal resistance to antimicrobials would be expected to provide insights into these mechanisms. In addition, MtrF (and/or its homologues) might have other important, as yet unknown, functions that will be elucidated with further study.
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Acknowledgements |
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Footnotes |
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