Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae

Wendy L. Veal1 and William M. Shafer1,2,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mtrCDE-encoded efflux pump of Neisseria gonorrhoeae provides gonococci with a mechanism to resist structurally diverse antimicrobial hydrophobic agents (HAs). Strains of N. gonorrhoeae that display hypersusceptibility to HAs often contain mutations in the efflux pump genes, mtrCDE. Such strains frequently contain a phenotypically suppressed mutation in mtrR, a gene that encodes a repressor (MtrR) of mtrCDE gene expression, and one that would normally result in HA resistance. We have recently examined HA-hypersusceptible clinical isolates of gonococci that contain such phenotypically suppressed mtrR mutations, in order to determine whether genes other than mtrCDE are involved in HA resistance. These studies led to the discovery of a gene that we have designated mtrF, located downstream of the mtrR gene, that is predicted to encode a 56.1 kDa cytoplasmic membrane protein containing 12 transmembrane domains. Expression of mtrF was enhanced in a strain deficient in MtrR production, indicating that this gene, together with the closely linked mtrCDE operon, is subject to MtrR-dependent transcriptional control. Orthologues of mtrF were identified in a number of diverse bacteria. Except for the AbgT protein of Escherichia coli, their products have been identified as hypothetical proteins with unknown function(s). Genetic evidence is presented that MtrF is important in the expression of high-level detergent resistance by gonococci. We propose that MtrF acts in conjunction with the MtrC–MtrD–MtrE efflux pump, to confer on gonococci high-level resistance to certain HAs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to a number of hydrophobic drugs, dyes and detergents, and antibacterial peptides in Neisseria gonorrhoeae is provided by an energy-dependent efflux pump termed Mtr, for multiple transferable resistance.17 The Mtr system is similar in organization to several other Gram-negative efflux systems, in which three proteins are believed to form a channel across the inner and outer membranes, through which antimicrobials are exported.810 In the case of the Mtr system, the three proteins are encoded by an operon, mtrCDE.2,46 The MtrC protein belongs to the membrane fusion protein (MFP) family11 and is presumed to link MtrD, the resistance/nodulation/division (RND) transporter11 located in the cytoplasmic membrane, with MtrE, an outer membrane protein that serves as a channel for export of antimicrobials to the extracellular fluid.3,5,6

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth conditions

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.


View this table:
[in this window]
[in a new window]
 
Table 1.  Mutations in mtrF suppress high-level resistance resulting from mtrR mutations
 
PCR amplification, DNA sequencing and analyses of sequence information

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 manufacturer’s 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 User’s 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 transcriptase–PCR (RT–PCR),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 manufacturer’s 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 manufacturer’s 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 SDS–PAGE33 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Discovery of mtrF

We15 demonstrated previously that the HA-hypersusceptible laboratory strains BR87 and BR54, of the FA19 genetic lineage,16 contain small (6–10 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-39->Ala-39) in the first helical domain of the helix–turn–helix 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).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 1. Location and organization of the mtr locus. (a) Organization of the region upstream of the mtrCDE operon showing ClaI restriction sites. Sizes of genes and intergenic regions are indicated above and below the line diagram. The indicated distance between mtrF and degR does not include the 33 bp region between the two potential start codons (shown in b). (b) Organization of the region upstream of mtrF. The putative –10 and –35 elements of the mtrF promoter are underlined and labelled. A potential ribosome binding site is underlined and indicated by RBS. The two potential ATG translational start codons are shown in bold; the second one is the designated initiation codon used throughout this report. The first 13 predicted amino acids of MtrF are indicated below the respective codons in italics.

 
Together with the mtrR DNA sequencing data, the Southern hybridization results suggested that transformant WV5 possesses DNA sequences from strain FA140 that have recombined at the mtr locus. An examination of the FA1090 genome sequence database, available online from the Gonococcal Genome Sequencing Project (www.genome.ou.edu) revealed that the relevant ClaI fragment was the appropriate size (3.3 kb) for one containing mtrC, mtrR, degR and part of another open reading frame, which we initially designated orfX, and then (see below) mtrF (Figure 1a). In the FA1090 genome sequence, there is another ClaI site in the orfX sequence 370 bp downstream from the one defining the end of the 3.3 kb fragment. It was thought possible, therefore, that a mutation had altered the first ClaI site within orfX in EU75, as observed. Sequencing of orfX from strains EU75, WV5 and FA19 revealed that EU75 indeed contained a mutation in the ClaI site (data not presented), as well as three other mutations that overall would result in four amino acid substitutions in the predicted protein (Figure 2a). Importantly, all of these mutations had been repaired in the HAR transformant strain WV5, restoring the predicted amino acid sequence to that of strain FA19 (Figure 2a).



View larger version (74K):
[in this window]
[in a new window]
 
Figure 2. (a) Predicted amino acid sequence (single letter code) of MtrF from strains FA19, EU75 and WV5. Underlined amino acids in the EU75 MtrF sequence highlight differences between it and the MtrF protein of strains FA19 and WV5. (b) Predicted topology of MtrF showing the location of mtrF-75 mutations. Topology map of MtrF demonstrating 12 transmembrane-spanning domains (shown as numbered ovals) generated using the TopPred II program.24 The nature and approximate locations of the four amino acid substitutions present in clinical isolate EU75 are indicated as wild-type FA19->EU75; the codon number of the change is indicated above the designation.

 
Inspection of the nucleotide sequence of orfX suggested two potential in-frame translational start codons (Figure 1b). We favour use of the second codon, because results from RT–PCR studies (data not presented) revealed that transcription was probably directed by a promoter element located 17 nucleotides upstream, with translation directed by a ribosome-binding site (RBS) positioned six nucleotides upstream of the ATG start codon (Figure 1b). The alternative translational start point (Figure 1b) lacks a credible RBS. Based on the use of the second ATG start codon, the product of orfX (GenBank accession no. AF176820) is predicted to be a 522 amino acid protein, with a molecular weight of 56.1 kDa, and a pI of 8.3. Using the TopPred II Program,24 we deduced that the orfX-encoded protein (subsequently termed MtrF) would be a CMP with 12 transmembrane domains (Figure 2b). Three of the amino acid substitutions in the protein produced by strain EU75 would be within putative transmembrane domains of the protein. The alanine to valine (codon 223) and alanine to threonine (codon 224) substitutions would be near the centre of the fifth predicted transmembrane domain, while the isoleucine to valine substitution (codon 281) would be within the sixth transmembrane region (Figure 2b). The fourth mutation, a leucine for isoleucine substitution (codon 192), would be located in the periplasmic loop between the fourth and fifth transmembrane domains (Figure 2b).

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 RT–PCR 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.



View larger version (36K):
[in this window]
[in a new window]
 
Figure 3. Transcription of rmp, mtrF and mtrD in the presence and absence of MtrR. RT–PCR was performed on RNA extracted from FA19 (wild-type) and KH15, which lacks mtrR expression due to a base pair deletion within the promoter region.12 DNA indicates a PCR control using chromosomal DNA from the same species. The – indicates a control reaction containing no reverse transcriptase to demonstrate that the RNA is free from DNA contamination. Expression of the rmp gene is used as a control for RNA amounts present, since it is not under the control of MtrR.

 
MtrF defines a new protein family

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.



View larger version (104K):
[in this window]
[in a new window]
 
Figure 4. Amino acid sequence alignment of MtrF and MtrF-like proteins from diverse bacteria. This alignment was prepared using DNAStar MegAlign by the Clustal method; complete alignment of the entire sequence has been omitted to conserve space. Motifs were identified with the aid of the MEME and MAST programs.26,27 Shaded boxes indicate residues that are identical to the alignment consensus sequence. Sequence numbers on the left refer to the position of the leftmost residues on each line. Bars indicate the conserved motif regions; the motif consensus sequence presented beneath each bar is displayed as follows: x, any amino acid; capital letters, the residue is conserved in >75% of the aligned sequences; lowercase letters, the residue is conserved in >40% of the aligned sequences. Abbreviations on the right correspond to the first initials of the genus and species of the sequence source, as listed in Table 2; C.d. refers to C. difficile.

 

View this table:
[in this window]
[in a new window]
 
Table 2.  MtrD and MtrF homologues in sequenced bacterial strains
 


View larger version (9K):
[in this window]
[in a new window]
 
Figure 5. Phylogenetic tree of full-length MtrF-like proteins. The unbalanced phylogenetic tree was generated by DNAStar MegAlign using the Clustal method. The scale beneath the diagram measures the distance between sequences; units indicate the number of substitution events. The branch distances correspond to sequence divergence. For E. coli and N. gonorrhoeae, the gene designation is indicated in parentheses.

 
The alignment analysis of MtrF, and similar proteins (Figure 4), revealed several highly conserved amino acid sequences among the proteins. With the aid of the MEME26 and MAST27 programs, five of these highly conserved sequences have been defined as motifs, indicated by brackets in Figure 4. These sequences are represented by amino acids 87–114, 165–186, 235–246, 362–377 and 413–465 of MtrF (Figure 4). Another potentially interesting region of the MtrF protein, and a possible sixth motif, is that between amino acids 218 and 225 (Figure 4). Although the amino acid identity is not as strong as those in the motifs mentioned previously, most of the variations from the NWFFXXAS pattern are conservative substitutions. Since the alanine found in this pattern, Ala-224, as well as the one preceding it in the MtrF sequence, Ala-223, are changed in EU75 to a valine and threonine (as shown in Figure 2), this putative motif may well be important to the function of the protein.

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.


View this table:
[in this window]
[in a new window]
 
Table 3.  Effects of mtrFmtrD double mutations on HA susceptibility
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of gonococcal resistance to structurally diverse HAs provided by the MtrC–MtrD–MtrE efflux pump is controlled transcriptionally by the MtrR repressor; increased resistance to HAs is thus conferred by mutations affecting MtrR structure, or mtrR expression.2,3,12,14 The significance of such mutations in vivo has been previously shown through the characterization of mtrR mutations in HAR clinical isolates.14,18,19 Here we have demonstrated that a novel protein, MtrF, is also involved in the expression of high-level HAR mediated by the mtr system. This was surmised from the ability of a non-polar insertional mutation in mtrF to reduce HA resistance, expressed by strain FA140. Interestingly, insertional mutation of mtrF in wild-type strain FA19, or in the HA-hypersusceptible strain BR54, which contains a nonsense mutation in mtrD, did not enhance the susceptibility of these strains to HAs. Moreover, we interpret the results of the genetic epistasis test to indicate that MtrF is not a core component of the pump. Rather, it seems to behave as an accessory factor, promoting full levels of resistance mediated by the MtrC–MtrD–MtrE pump, when gonococci genetically capable of expressing HAR are exposed to high levels of HAs. We propose, therefore, that MtrF is located in the inner membrane, close to the MtrC–MtrD–MtrE efflux pump protein complex. We do not believe that MtrF, by itself, can mediate HA resistance, because its expression in E. coli does not change the susceptibility of this bacterium to antimicrobials (data not presented). Furthermore, gonococcal strains (e.g. strain BR54), with mutations in the mtrCDE operon, remain highly HA susceptible. In addition, it is unlikely that loss of mtrF decreases resistance by disrupting transport of secreted proteins, because inactivation of mtrF, in a ß-lactamase-producing strain, does not alter the level of penicillin resistance, indicating that ß-lactamase secretion is unaffected (data not presented). We do not yet know whether MtrF performs a structural role by transiently interacting with pump proteins, for example, to stabilize the complex in some way. Alternatively, it may be involved in delivering excess HAs to the pump for subsequent export, or in energy transduction needed for increased HA resistance.

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 (80–100%) identity to MtrF, while cluster II accommodates those with much lower percentage identities (36–48%). 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.


    Acknowledgements
 
We thank J. Balthazar and A. Yellen for excellent technical assistance, D. Cox for assistance in figure preparation, B. Nichols for E. coli strains, Stephen A. Morse (Centers for Disease Control and Prevention, Atlanta, GA, USA) for providing strain EU75 and P. F. Sparling (Department of Medicine, Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC, USA) for strains FA19 and FA140. This work was supported by a grant from the National Institutes of Health AI-21150 (W.M.S.). W.L.V. was supported in part by NIH Training Grant 5 T32 A107470-04. W.M.S. was supported by a Senior Research Career Scientist Award from the Veterans Affairs Medical Research Service. We would like to express our appreciation of all sequencing projects for early data release used in homology comparisons. Thanks to D. Dyer, B. Roe and the staff of the University of Oklahoma’s Advanced Center for Genome Technology (Norman OK, USA) for placing rapidly the existing FA1090 genomic sequence information on the World Wide Web. The sequencing of the FA1090 genome is supported by PHS grant AI38399 (D. Dyer and B. Roe) from the NIH. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org; sequencing of C. crescentus and S. putrefaciens was accomplished with support from the Department of Energy (DOE); sequencing of V. cholerae and S. aureus COL was accomplished with support from the National Institute of Allergy and Infectious Diseases (NIAID) and S. aureus with support from the Merck Genome Research Institute (MGRI). Sequencing groups at the Sanger Centre also produced sequence data; sequencing of Y. pestis and C. difficile was supported by Beowulf Genomics. A preliminary account of these studies was presented at the Twelfth International Pathogenic Neisseria Conference held in Galveston, TX, USA, 12–17 November 2000.


    Footnotes
 
* Corresponding author. Tel: +1-404-728-7688; Fax: +1-404-329-2210; E-mail: wshafer{at}emory.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Maness, M. J. & Sparling, P. F. (1973). Multiple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae. Journal of Infectious Diseases 128, 321–30.[ISI][Medline]

2 . Pan, W. & Spratt, B. G. (1994). Regulation of the permeability of the gonococcal cell envelope by the mtr system. Molecular Microbiology 11, 769–75.[ISI][Medline]

3 . Hagman, K. E., Pan, W., Spratt, B. G., Balthazar, J. T., Judd, R. C. & Shafer, W. M. (1995). Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141, 611–22.[Abstract]

4 . Lucas, C. E., Hagman, K. E., Levin, J. C., Stein, D. C. & Shafer, W. M. (1995). Importance of lipooligosaccharide structure in determining gonococcal resistance to hydrophobic antimicrobial agents resulting from the mtr efflux system. Molecular Microbiology 16, 1001–9.[CrossRef][ISI][Medline]

5 . Delahay, R. M., Robertson, B. D., Balthazar, J. T., Shafer, W. M. & Ison, C. A. (1997). Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents. Microbiology 143, 2127–33.[Abstract]

6 . Hagman, K. E., Lucas, C. E., Balthazar, J. T., Snyder, L., Nilles, M., Judd, R. C. et al. (1997). The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system. Microbiology 143, 2117–25.[Abstract]

7 . Shafer, W. M., Qu, X.-D., Waring, A. J. & Lehrer, R. I. (1998). Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family. Proceedings of the National Acadamy of Sciences, USA 95, 1829–33.[Abstract/Free Full Text]

8 . Nikaido, H. (1996). Multidrug efflux pumps of Gram-negative bacteria. Journal of Bacteriology 178, 5853–9.[Free Full Text]

9 . Nikaido, H. (1998). Antibiotic resistance caused by gram-negative multidrug efflux pumps. Clinical Infectious Diseases 27, Suppl. 1, S32–41.[ISI][Medline]

10 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiological Reviews 60, 575–608.[Abstract]

11 . Saier, M. H., Jr, Tam, R., Reizer, A. & Reizer, J. (1994). Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Molecular Microbiology 11, 841–7.[ISI][Medline]

12 . Hagman, K. E. & Shafer, W. M. (1995). Transcriptional control of the mtr efflux system of Neisseria gonorrhoeae. Journal of Bacteriology 177, 4162–5.[Abstract]

13 . Lucas, C. E., Balthazar, J. T., Hagman, K. E. & Shafer, W. M. (1997). The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae. Journal of Bacteriology 179, 4123–8.[Abstract]

14 . Shafer, W. M., Balthazar, J. T., Hagman, K. E. & Morse, S. A. (1995). Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids. Microbiology 141, 907–11.[Abstract]

15 . Veal, W. L., Yellen, A., Balthazar, J. T., Pan, W., Spratt, B. G. & Shafer, W. M. (1998). Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae. Microbiology 144, 621–7.[Abstract]

16 . Sparling, P. F., Sarubbi, F. A. & Blackman, E. (1975). Inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae. Journal of Bacteriology 124, 740–9.[ISI][Medline]

17 . Eisenstein, B. I. & Sparling, P. F. (1978). Mutations to increased antibiotic sensitivity in naturally-occurring gonococci. Nature 271, 242–4.[ISI][Medline]

18 . Xia, M., Whittington, W. L. H., Shafer, W. M. & Holmes, K. K. (2000). Gonorrhea among men who have sex with men: Outbreak caused by a single genotype of erythromycin-resistant Neisseria gonorrhoeae with a single base pair deletion in the mtr promoter region. Journal of Infectious Diseases 181, 280–2.

19 . Zarontonelli, L., Borthagaray, G., Lee, E.-H. & Shafer, W. M. (1999). Decreased azithromycin susceptibility of Neisseria gonorrhoeae due to mtrR mutations. Antimicrobial Agents and Chemotherapy 43, 2468–72.[Abstract/Free Full Text]

20 . Zarontonelli, L., Borthagaray, G., Lee, E.-H., Veal, W. L. & Shafer, W. M. (2001). Decreased susceptibility to azithromycin and erythromycin mediated by a novel mtrR promoter mutation in Neisseria gonorrhoeae. Journal of Antimicrobial Chemotherapy 47, 651–4.[Abstract/Free Full Text]

21 . Shafer, W. M., Guymon, L. F., Lind, I. & Sparling, P. F. (1984). Identification of an envelope mutation (env-10) resulting in increased antibiotic susceptibility and pyocin resistance in a clinical isolate of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 25, 767–9.[ISI][Medline]

22 . McAllister, C. F. & Stephens, D. S. (1993). Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family. Molecular Microbiology 10, 13–24.[ISI][Medline]

23 . von Heijne, G. (1992). Membrane protein structure prediction: hydrophobicity analysis and the positive-inside rule. Journal of Molecular Biology 225, 487–94.[ISI][Medline]

24 . Claros, M. G. & von Heijne, G. (1994). TopPredII: an improved software for membrane protein structure predictions. CABIOS 10, 685–96.[Medline]

25 . Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.[Abstract/Free Full Text]

26 . Bailey, T. L. & Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. In Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28–36. AAAI Press, Menlo Park, CA, USA.

27 . Bailey, T. L. and Gribskov, M. (1998). Combining evidence using p-values: application to sequence homology searches. Bioinformatics 14, 48–54.[Abstract]

28 . Gunn, J. S. & Stein, D. C. (1996). Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae. Molecular and General Genetics 251, 509–17.[CrossRef][Medline]

29 . Ménard, R., Sansonetti, P. J. & Parsot, C. (1993). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC and IpaD as effectors of Shigella flexneri entry into epithelial cells. Journal of Bacteriology 175, 5899–906.[Abstract]

30 . Biran, D., Brot, N., Weissbach, H. & Ron, E. Z. (1995). Heat shock-dependent transcriptional activation of the metA gene of Escherichia coli. Journal of Bacteriology 177, 1374–9.[Abstract]

31 . Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. et al. (1992). Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY, USA.

32 . Clark, V. L., Campbell, L. A., Palermo, D. A., Evans, T. M. & Klimpel, K. W. (1987). Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae. Infection and Immunity 55, 1359–64.[ISI][Medline]

33 . Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5.[ISI][Medline]

34 . Hussein, M. J., Green, J. M. & Nichols, B. P. (1998). Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli. Journal of Bacteriology 180, 6260–8. [Abstract/Free Full Text]