JOURNAL OF BACTERIOLOGY, Jan. 2009, p. 287–297
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 1
MtrR Modulates rpoH Expression and Levels of Antimicrobial
Resistance in Neisseria gonorrhoeae?
Jason P. Folster,1,2† Paul J. T. Johnson,1,2† Lydgia Jackson,3,4† Vijaya Dhulipali,1,2
David W. Dyer,3,4and William M. Shafer1,2*
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 303221; Laboratories of
Bacterial Pathogenesis, VA Medical Center, Decatur, Georgia 300332; and Laboratory for Genomics and
Bioinformatics3and Department of Microbiology and Immunology, University of
Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731044
Received 18 August 2008/Accepted 21 October 2008
The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which
encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci
to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to deter-
mine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome
microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive
and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject
to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general
stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by
MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH
promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely
indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress
rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H2O2) and an antibiotic
(erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability
to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gono-
coccal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability
of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal
susceptibility to H2O2.
Neisseria gonorrhoeae is a gram-negative strict human patho-
gen and the causative agent of the sexually transmitted disease
gonorrhea. Worldwide, over 60 million cases of gonorrhea
occur each year (7, 33). Although the incidence of disease in
the United States has decreased since the 1970s, there has
since been an increase in antibiotic-resistant strains reported in
the United States and elsewhere (25, 33). Indeed, the preva-
lence of gonococci resistant to penicillin, tetracycline, and/or
fluoroquinolones became so significant over the past 25 years
that in 2007 the Centers for Disease Control and Prevention
added N. gonorrhoeae to the list of “superbugs”.
The capacity of gonococci and other bacteria to employ drug
efflux pumps to recognize and export antibiotics has attracted
considerable attention, as these pumps can impact the efficacy
of antibiotic therapy of infections (18, 19, 25, 30, 34, 35).
Moreover, since certain efflux pumps recognize host-derived
antimicrobial agents, such as antimicrobial peptides (24), there
is increasing suspicion that they provide bacteria with a mech-
anism to escape innate host defenses (13, 21). The gonococcal
MtrC-MtrD-MtrE efflux pump can export a variety of antibi-
otics (17, 28), including penicillin (30) and macrolides (35),
and antimicrobial peptides, such as human LL-37 (24).
The mtrCDE operon is negatively regulated by the MtrR
repressor (10, 16, 23), which is encoded by a gene (mtrR)
immediately upstream of, but transcriptionally divergent from,
mtrCDE (19). Two homodimers of MtrR bind to a DNA se-
quence within the mtrCDE promoter (12). Point mutations in
the MtrR-binding site (16), a single-base-pair deletion within a
13-bp inverted-repeat sequence in the mtrR promoter (10), or
missense mutations that cause radical amino acid replacements
within the helix-turn-helix motif of MtrR all can result in de-
repression of mtrCDE expression (23). Derepression of
mtrCDE results in decreased antimicrobial susceptibility of
gonococci (10, 11, 23) and increased fitness of gonococci in an
experimental murine lower genital tract infection model (31).
Consequently, it is important to define the regulatory prop-
erties of MtrR. DNA-binding proteins such as MtrR that neg-
atively regulate bacterial efflux pump genes have been consid-
ered to be “local” gene regulators, although there is increasing
evidence that they can directly or indirectly influence the ex-
pression of other genes (6, 15). In order to define the genes in
gonococci regulated by MtrR, we employed a microarray anal-
ysis of total RNA extracted from mid-logarithmic-phase broth
cultures of isogenic MtrR-positive and MtrR-negative gono-
cocci. Through this analysis, we found that MtrR can directly
or indirectly regulate 69 genes, which are distributed through-
out the genome. We identified 47 MtrR-repressed genes (in-
cluding the previously described mtrCDE operon) and 22
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Emory University School of Medicine, At-
lanta, GA 30322. Phone: (404) 728-7688. Fax: (404) 329-2210. E-mail:
† J.P.F., P.J.T.J., and L.J. contributed equally to this study.
?Published ahead of print on 31 October 2008.
MtrR-activated genes. An MtrR-repressed gene of particular
interest, which was the subject of this investigation, was rpoH.
rpoH encodes the alternative stress response sigma factor
RpoH (sigma 32), which appears to be essential for gonococcal
viability even under normal growth conditions (3, 8). Earlier
studies showed that rpoH expression is increased during expo-
sure of gonococci to certain stress conditions, such as growth at
elevated temperatures (8), and during bacterial contact with
cultured epithelial cells (2, 3). Moreover, certain RpoH-regu-
lated genes are differentially expressed during exposure of
gonococci to H2O2(29). Thus, apart from its regulation of
mtrCDE and levels of gonococcal resistance to hydrophobic
antimicrobial agents (20, 21, 24, 30), MtrR regulation of rpoH
expression may have significance for gonococcal survival in
vivo, particularly at sites rich in neutrophils producing large
quantities of H2O2.
MATERIALS AND METHODS
Bacterial strains, plasmids, oligonucleotides, and culture conditions. The
bacterial strains used in this study are listed in Table 1. Escherichia coli strain
TOP10 (Invitrogen, Carlsbad, CA) or strain DH5? mcr was used in all cloning
experiments. E. coli strains were grown in Luria-Bertani broth or on Luria-
Bertani agar plates at 37°C. N. gonorrhoeae strain FA19 was used as the primary
gonococcal strain (22). N. gonorrhoeae strains were grown on gonococcal medium
base (GCB) agar (Difco Laboratories, Detroit, MI) containing glucose and iron
supplements (22) at 37°C under 3.8% (vol/vol) CO2or in GCB broth with
supplements and sodium bicarbonate as described previously (22). The plasmids
and oligonucleotide primers used in this investigation are listed in Tables 1 and
2. All chemicals were purchased from Sigma Biochemical (St. Louis, MO).
RNA isolation. RNA was isolated from 50-ml GCB broth cultures of strains
FA19 and JF1 (the same as FA19 but with mtrR deleted [?mtrR] ) grown to
mid-log phase using a hot-phenol method as previously described (4). Samples
were then DNase treated on column (Qiagen DNase kit), and the RNA was
recovered using the Qiagen RNeasy Mini kit. RNA was quantified by Nano-
Drop1000 (NanoDrop Technolgies), and the RNA integrity was analyzed on an
Agilent bioanalyzer (Agilent Technologies). The RNA samples were stored at
?80°C until further use.
A custom multipathogen
MPAUT1a520274F, was utilized for the microarray experiments in the present
study. Probe pairs were designed for each gene, consisting of 11 probe pairs (22
features) with a perfect match and a single-nucleotide mismatch. The N. gonor-
rhoeae genome probe set (1,925 genes) was represented on the array.
TABLE 1. Gonococcal strains and plasmids used
Strain or plasmidRelevant genotype or remarks
Like FA19 but ?mtrR
Like JF1 but mtrR?a
Like JF1 but mtrR?brpoH-lacZ
Like FA19 but rpoH-lacZ
Like JF1 but rpoH-lacZ
Like JF6 but rpoH-lacZ
Like FA19 but grpE-lacZ
Like JF1 but grpE-lacZ
Like JF6 but grpE-lacZ
Like FA19 but rpoH1-lacZc
Like JF1 but rpoH1-lacZc
Like FA19 but mtrR-lacZ
P. F. Sparling
pLES94Cloning vector containing promoterless lacZ for insertion of gonococcal genes
between proA and proB (27)
NICS vector (25) used for insertion of mtrR between lctP and aspC
Same as pGCC3 with IPTG-inducible lacZ promoter
Like pGCC3 but mtrR?
Like pGCC4 but mtrR?
amtrR from pJF1 is under the control of its own promoter in this strain.
bmtrR from pJF2 is under the control of the lacZ promoter in this strain.
cThis fusion lacks the 172-bp rpoH leader sequence.
TABLE 2. Oligonucleotides used
288FOLSTER ET AL.J. BACTERIOL.
cDNA labeling and hybridization. Sample preparation and hybridization were
done at the University of Iowa DNA Facility (http://dna-9.int-med.uiowa.edu/)
according to the Affymetrix Genechip Expression Analysis technical manual
prokaryotic protocol. Briefly, biotinylated cDNA was generated from 10 ?g of
total RNA and fragmented prior to hybridization to the custom chip at 45°C for
16 h. The arrays were stained using streptavidin-phycoerythrin (ProkGE-
WS2v3_450 protocol; Affymetrix, Inc.), and the scanned images (Affymetrix
GeneChip Scanner 3000) were processed using Genechip Operating Software
version 1.4 (Affymetix, Inc.). Hybridizations were done on three separate bio-
logical replicates of each isolate.
Array analysis. Data files generated by Genechip Operating Software (Af-
fymetix, Inc.) were imported into GeneSpring GX 7.3.1 (Agilent Technologies)
and normalized per chip by dividing each measurement by the 50th percentile of
all the measurements in that sample and per gene by dividing each gene by the
median of its measurements across all samples. The cross-gene error model was
operating based on replicates. The normalized data from all samples were fil-
tered on genes flagged as present or marginal, with the resulting gene list used
for further gene expression analysis and clustering. Change was expressed as the
ratio of gene expression of the wild-type FA19 over the same gene expression for
the mutant. Genes with a differential expression of ?1.5-fold and a P value of
0.05 were selected.
Construction of mtrR-complemented strains, inducible expression of mtrR,
detection of MtrR, and antimicrobial susceptibility in induced cultures. The
construction of pJF1, which contains the wild-type mtrR coding sequence and
promoter sequence from strain FA19 cloned into pGCC3 (27), and strain JF6
(like JF1 but with this mtrR sequence, including its promoter, located between
aspC and lctP) has been described by Folster and Shafer (5). In order to engineer
an IPTG (isopropyl-?-D-thiogalactopyranoside)-inducible copy of mtrR, the mtrR
coding sequence with its ribosome binding site was PCR amplified from chro-
mosomal DNA prepared from wild-type strain FA19 using primers 5?mtrR and
3?mtrR (Table 2). The resulting DNA fragment was inserted into the PmeI and
PacI sites of pGCC4, which contains the lacZ promoter (27) (kindly provided by
A. Jerse and H. Seifert) to produce pJF2, and the correct orientation and
nucleotide sequence were confirmed by DNA sequencing. pJF2 was digested
with ClaI, and the fragment containing the gonococcal lctP gene, mtrR, ermC (an
erythromycin [Ery] resistance cassette), and aspC was purified and used to
transform gonococcal strain FA19 ?mtrR. Transformations were performed as
previously described (9). Transformants were selected on GCB agar containing
1 ?g/ml of Ery, and the resulting strain was named JF7 (like JF1, but mtrR?).
For inducible expression of mtrR, strain JF7 was grown on GCB agar as
described above. The overnight growth was then used to inoculate 50 ml of GCB
broth, and the culture was grown at 37°C with shaking (200 rpm) until the optical
density at 600 nm reached 0.2, at which time it was split into two equal samples.
One sample received IPTG (1 mM), while the other received an equal volume of
sterile distilled water. These cultures were grown for 1 h, and samples were then
harvested by centrifugation. MtrR was detected in solubilized cell extracts by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
Western immunoblotting as described previously (6). MtrR was detected using a
rabbit anti-MtrR antiserum (1:5,000 dilution) prepared by Protein Tech Group,
Inc. (Chicago, IL), with purified MtrR fused to maltose-binding protein (MBP)
(15) as the antigen and was visualized on the blot using goat anti-rabbit immu-
noglobulin G coupled to alkaline phosphatase (1:5,000 dilution). IPTG-induced
and control samples were also evaluated for their susceptibility to Ery (0.5 ?g/ml)
by an efficiency-of-plating assay described previously (22), using agar plates with
or without 1 mM IPTG. H2O2susceptibility was determined using a disk diffu-
sion assay containing filter disks (1 cm) presoaked in 3% (vol/vol) H2O2that
were placed on freshly inoculated GCB agar plates that contained or lacked 1
Construction and analysis of rpoH-lacZ, grpE-lacZ, and mtrR-lacZ fusions.
Translational lacZ fusions were constructed as previously described (5, 6). In
brief, the promoter sequence of rpoH was amplified by PCR from strain FA19
using primers 5?PrpoH and 3?PrpoHB (Table 2). The resulting DNA fragment
was inserted into the BamHI site of pLES94 (26), and the recombinant plasmid
was introduced into E. coli DH5? mcr by transformation. The correct orientation
of the insertion was confirmed by PCR analysis and DNA sequencing. The
plasmid was used to transform strains FA19, JF1, and JF6 to allow insertion into
the chromosomal proAB gene, and transformants were selected on GCB agar
containing 1 ?g/ml of chloramphenicol. Representative transformants obtained
with the three recipient strains were termed JF8, JF9, and JF10, respectively
(Table 1). Strains bearing a grpE-lacZ fusion were prepared essentially as de-
scribed above, but the oligonucleotide primers 5?PgrpE and 3?PgrpE (Table 2)
were used to PCR amplify a 300-bp fragment encompassing the upstream se-
quence of grpE and the first two codons. The strains bearing the grpE-lacZ fusion
were termed JF11 (FA19 grpE-lacZ), JF12 (FA19 ?mtrR grpE-lacZ), and JF13
(FA19 ?mtrR mtrR?grpE-lacZ), respectively (Table 1). For construction of an
mtrR-lacZ fusion in strain FA19, oligonucleotide primers mtrC1 and pmtrR
(Table 2) were used to PCR amplify mtrR; the strain bearing the mtrR-lacZ
fusion was termed JF16 (FA19 mtrR-lacZ). ?-Galactosidase (?-Gal) assays were
conducted as previously described (5, 6).
Deletion of the rpoH extended leader sequence. PCR mutagenesis was used to
delete the 172-bp rpoH leader sequence and was performed using the overlap-
ping primers 5??PrpoH and 3??PrpoH (Table 2). First, two fragments were
amplified by PCR from FA19 chromosomal DNA using the primer set pairs
5?PrpoH/3??PrpoH and 3?PrpoHB/5??PrpoH. The resultant fragments were
purified after agarose gel electrophoresis using the Qiaquick purification kit
(Qiagen Inc., Valencia, CA), and they then served as both primers and templates
for a second PCR. After eight reaction cycles, primers 5?PrpoH and 3?PrpoHB
were added to the PCR mixture, and amplification continued for an additional 25
cycles. The resulting DNA fragment containing the deletion was purified and
served as the template for the last PCR using primers 5?PrpoH and 3?PrpoHB.
The resulting DNA fragment was inserted into the BamHI site of pLES94,
resulting in the rpoH?-lacZ construct. The recombinant plasmid was introduced
into DH5? mcr by transformation. Correct insertion and orientation were con-
firmed by PCR analysis, and DNA-sequencing analysis confirmed the mutation
of the MtrR-binding site (data not presented). The plasmid was used to trans-
form strains FA19 and JF1 to allow insertion into the chromosomal proAB gene.
Transformants were selected on GCB agar containing 1 ?g/ml of chloramphen-
icol; representative transformants obtained with recipient strains FA19 and JF1
were termed JF14 and JF15, respectively (Table 1).
EMSA and DNase I protection. Electrophoretic mobility shift assays (EMSAs)
using purified MBP fused to MtrR were performed as previously described (5, 6,
15). In brief, the 184-bp promoter region of rpoH lacking the leader sequence
was amplified by PCR using 5?PrpoH and 3?PrpoHB from strain JF14 (Table 1).
The 184-bp nonspecific probe was PCR amplified from FA19 chromosomal
DNA using primers 5??PrpoH and 3?PrpoHB (Table 2). The resulting PCR
products were end labeled with [?-32P]dATP using T4 polynucleotide kinase
(New England Biolabs, Beverly, MA). The labeled DNA fragments (10 ng) were
incubated with purified MtrR in 30 ?l of reaction buffer [10 mM Tris-HCl (pH
7.5), 0.5 mM dithiothreitol, 0.5 mM EDTA, 4% (vol/vol) glycerol, 1 mM MgCl2,
50 mM NaCl, poly(dI-dC) (0.05 ?g/ml), salmon sperm (200 ng/ml)] at 4°C for 20
min. Samples were subjected to electrophoresis on a 6% (wt/vol) polyacrylamide
gel at 4°C, followed by autoradiography.
DNase I protection assays were performed as previously described (16). Target
DNA sequences for DNase I footprints were generated by PCR using the oligo-
nucleotide primers 5?PrpoH and 3?PrpoHB (Table 2) to generate a 184-bp
promoter region of rpoH lacking the leader sequence from strain JF14. Target
DNA was labeled at the 5? end of one strand as described for EMSA. Purified
MBP-MtrR protein was allowed to bind in the binding buffer as in the EMSA
before CaCl2and MgCl2were added to final concentrations of 2.5 mM and 5
mM, respectively. Five units of DNase I (Promega, Madison, WI) were then
added to the reaction mixture, which was incubated at room temperature for 1
min. Digestion was stopped by the addition of NaCl to 250 mM, and the reaction
mixture was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) before
precipitation with 100% ethanol for 30 min at ?20°C. The pellet was washed four
times with 70% (vol/vol) ethanol, vacuum dried, and resuspended in gel loading
buffer, which consisted of 0.1 M NaOH-formamide (1:2 [vol/vol]), 0.1% (vol/vol)
xylene cyanol, and 0.1% (vol/vol) bromophenol blue. Regions protected from
DNase I digestion were resolved on a 6% denaturing polyacrylamide gel, which
was dried and exposed to X-ray film for autoradiography.
Microarray data expression number. Gene expression data for all microarray
experiments can be retrieved from the Gene Expression Omnibus (GEO) data-
base at NCBI (http://www.ncbi.nih.gov/geo/) (accession number GSE12686).
RESULTS AND DISCUSSION
Identification of MtrR-regulated genes in gonococci. To
identify MtrR-regulated genes in N. gonorrhoeae, a microarray
analysis was performed that permitted a comparison of gono-
coccal transcript levels in total RNA prepared from mid-loga-
rithmic-phase cultures of wild-type strain FA19 and its isogenic
mtrR deletion mutant, strain JF1 (Table 1). Using an expres-
sion difference of 1.5-fold (P ? 0.05), analysis of the microar-
ray results allowed us to establish an MtrR regulon that at a
VOL. 191, 2009MtrR REGULATION OF rpoH289
minimum consists of 47 MtrR-repressed and 22 MtrR-acti-
vated genes (Table 3); these MtrR-regulated genes were dis-
tributed throughout the genome (Fig. 1). Four previously iden-
tified MtrR-repressed genes (mtrC, mtrD, mtrE, and mtrF)
were confirmed as being MtrR repressed by the array, which
validated the array as a means to identify MtrR-regulated
genes. Based on the annotation of the FA1090 genome se-
quence (GenBank accession no. AE004969), the majority of
MtrR-regulated genes were predicted to encode hypothetical
proteins, but several genes encoding proteins with known or
TABLE 3. MtrR-regulated genesain N. gonorrhoeae
GeneCommon name Change (fold) Functional classification
Putative ABC-type polyamine transporter
Alternative sigma factor
Pyrodoxamine 5?-phosphate oxidase
Putative prolipoprotein diacylglycerol transferase
Unknown (putative phage associated)
Heat shock chaperone
Heat shock chaperonin
MarR family regulator
AsnC family transcriptional regulator
Mtr efflux pump protein component
Mtr efflux pump protein component
Mtr efflux pump protein component
Mtr efflux pump protein component
Subunit F, NADH-quinone reductase
Heat shock protein
Putative periplasmic polyamine binding protein
NADH dehydrogenase subunit M
NADH dehydrogenase kappa subunit
NADH dehydrogenase I chain J
NADH dehydrogenase subunit C
Putative NADH dehydrogenase I chain A
Putative glutamate permease
Putative ABC-type polyamine transporter
Putative ATP-binding protein
Amino acid periplasmic binding protein
Putative RNase G/cytoplasmic axial filament protein
Unknown (putative phage associated)
Putative DNA helicase
Unknown (phage associated)
Glutamine synthetase adenylyltransferase
aGenes of interest to this investigation are highlighted in boldface.
290 FOLSTER ET AL. J. BACTERIOL.
putative functions were identified (Table 3). In particular, we
noted that expression of rpoH, which encodes the stress-related
sigma factor RpoH, and some RpoH-regulated genes (grpE,
clpB, and marR [8, 29]) were increased in strain JF1 relative to
the MtrR-positive parental strain FA19, suggesting that they
are MtrR repressed. Since RpoH expression in gonococci is
increased during exposure to certain environmental stresses
(8) when gonococci bind to cervical epithelial cells (2, 3) and is
essential for viability (3, 8), we studied the ability of MtrR to
regulate rpoH expression in N. gonorrhoeae.
Expression of rpoH and certain RpoH-regulated genes is
repressed by MtrR. To study the capacity of MtrR to regulate
the expression of rpoH, a translational lacZ reporter fusion
system was engineered. This translational fusion consisted of
365 bp of DNA sequence upstream of rpoH, which included
the promoter region (14), the first two codons of rpoH (Fig. 2),
and a promoterless lacZ gene. This construct was transformed
into isogenic strains FA19 and JF1 to create the transformant
strains JF8 (FA19 rpoH-lacZ) and JF9 (FA19 ?mtrR rpoH-
lacZ), respectively, resulting in a single copy of the rpoH pro-
moter fused translationally to lacZ within the proAB chromo-
somal locus. ?-Gal activity in cell lysates from these strains
indicated that expression of rpoH increased more than eight-
fold in the MtrR-negative strain JF9 compared to the MtrR-
positive strain JF8 (FA19 rpoH-lacZ) (Fig. 3A). To confirm
that these results were due to deletion of mtrR and not a polar
effect on other genes, strain JF9 was complemented with the
wild-type mtrR gene from strain FA19, which was inserted at a
secondary site within the gonococcal chromosome (between
the lctP and aspC genes) to create strain JF10 (FA19 ?mtrR
mtrR?rpoH-lacZ) (Table 1). Using this strain, we found that
complementation of the mtrR deletion in JF9 (FA19 ?mtrR
rpoH-lacZ) with the wild-type mtrR gene restored rpoH repres-
sion to a level similar to that of JF8 (like FA19, but rpoH-lacZ)
The sequence of the gonococcal rpoH promoter, as well as
the transcriptional start point and ribosome binding site, were
defined previously (8) (Fig. 2). This region contains an ex-
tended 172-bp RNA leader sequence, which was previously
observed to be involved in rpoH repression during growth
under normal conditions (14). To determine if the leader se-
quence was involved in MtrR repression of rpoH, an internal
deletion was engineered within the rpoH-lacZ construct and
transformed into strains FA19 and JF1 (FA19 ?mtrR) to create
strains JF14 (FA19 rpoH-lacZ) and JF15 (FA19 ?mtrR rpoH-
lacZ) (Table 1), respectively, and expression was measured.
We found that rpoH-lacZ expression from the wild-type pro-
moter and the promoter with the leader deleted did not differ
in either the MtrR-positive (JF14) or MtrR-negative (JF15)
background (Fig. 3B), indicating that MtrR-mediated repres-
sion of rpoH is independent of the leader sequence.
Gunesekere et al. (8) identified a panel of RpoH-regulated
genes in gonococci, and Stohl et al. (29) showed that some of
these were upregulated during bacterial exposure to H2O2.
Since our microarray analysis (Table 3) suggested that a subset
of these RpoH-regulated genes (clpB, grpE, and marR) is also
FIG. 1. Chromosomal-map positions of MtrR-regulated genes. The positions of MtrR-regulated genes in strain FA19 that were identified by
the microarray analysis (Table 3) are shown on the circular map of the gonococcal chromosome (strain FA1090). MtrR-activated genes are shown
in red, while MtrR-repressed genes are shown in green.
VOL. 191, 2009MtrR REGULATION OF rpoH291
subject to MtrR regulation, we examined the ability of MtrR to
control the expression of RpoH-regulated genes in gonococci
and used grpE for this purpose. We examined its expression in
isogenic MtrR-positive and MtrR-negative gonococci utilizing
a grpE-lacZ construct introduced into strains FA19, JF1 (FA19
?mtrR), and JF6 (FA19 ?mtrR mtrR?); the transformed strains
were termed JF11, JF12, and JF13, respectively (Table 1). The
results indicated that expression of grpE (Fig. 4) was elevated
in the absence of MtrR (see strain JF12) and restored to
near-wild-type levels (strain JF11) by complementation with
the intact mtrR gene (strain JF13).
As expression of RpoH-regulated grpE is induced during
exposure of gonococci to low levels of H2O2(29), we next
evaluated whether expression of mtrR, rpoH, and grpE was
altered when gonococci were exposed to the oxidizing agent.
Using the lacZ fusion strains JF8 (FA19 rpoH-lacZ), JF11
FIG. 2. Nucleotide sequence upstream of rpoH and identification of the MtrR-binding site. The nucleotide sequence of the DNA
upstream of rpoH is shown with the 172-bp leader sequence indicated by the dashed lines; the first two codons encoding methionine (M) and
asparagine (N) are also shown in boldface. The position of the transcriptional start point (8) is shown by the boldface A nucleotide
downstream of the ?10 sequence. The ?10 and ?35 sequences of the rpoH promoter identified previously (8) are shown with the ?10 and
?35 hexamers and are highlighted by a single line under the sequence. The 4-bp ribosome binding site (RBS) sequence is shown by an
underline. The MtrR-binding site identified by DNase I protection (Fig. 6) is shown by the wide bars above the coding strand and below the
noncoding strand. The boxed region shows a 15-nucleotide sequence on the coding strand (5?-GGC[-]CGTTTACATACA-3?) that is 73.3%
identical (the identical nucleotides are boldface in above sequence) to the MtrR-binding sequence (5?-GGCACGTTAGCACATA-3?)
upstream of mtrCDE on the noncoding strand (12); the hyphen in the sequence above represents a single-nucleotide gap in the rpoH
sequence compared to the mtrCDE bound by MtrR.
FIG. 3. MtrR regulation of rpoH expression. (A) Levels of ?-Gal activity (specific activity expressed as nanomoles of o-nitrophenyl-?-D-
galactopyranoside hydrolyzed per mg of protein) in strains JF8 (like FA19, but rpoH-lacZ), JF9 (like JF1, but rpoH-lacZ), and JF10 (like JF6 [Table
1], but rpoH-lacZ). Complementation of the mtrR deletion mutation resulted in reduced rpoH expression. The results are shown as average values
(? standard deviations) from three independent assays. The difference in ?-Gal activities between strains JF8 and JF9 was significant (P ? 0.001),
as was that between JF9 and JF10 (P ? 0.001). (B) MtrR repression of rpoH was independent of the 172-bp leader sequence upstream of rpoH,
as shown by similar levels of ?-Gal production between isogenic pairs (MtrR-positive strains JF8 and JF14 and MtrR-negative strains JF9 and
JF15) with (strains JF8 and JF9) or without (JF14 and JF15) the 172-bp leader sequence. The differences in ?-Gal activities between strains JF8
and JF14 and between JF9 and JF15 were not statistically significant.
292 FOLSTER ET AL. J. BACTERIOL.
(FA19 grpE-lacZ), and JF16 (FA19 mtrR-lacZ) (Table 1), we
monitored the expression of rpoH, grpE, and mtrR during a
brief (15-min) exposure of gonococci to 1.5 mM H2O2; this
concentration of H2O2did not reduce viability (data not pre-
sented). As is shown in Fig. 5, we found that under these
conditions, expression of both rpoH and grpE was increased
significantly (P ? 0.0001 and 0.002, respectively), while mtrR
expression was slightly but significantly (P ? 0.049) dampened.
In order to determine if H2O2induction of rpoH expression
would be enhanced in the absence of MtrR, we next compared
its expression when strains JF8 (FA19 rpoH-lacZ) and JF9 (like
JF8, but ?mtrR) were exposed to 1.0 mM H2O2for 15 min.
Although rpoH expression was nearly 15-fold greater in strain
JF9 than in JF8, the changes in expression after H2O2exposure
were nearly identical (data not presented). We propose that
although MtrR can control constitutive levels of rpoH and grpE
exposure, it is not necessarily involved in their H2O2-inducible
expression. Alternatively, under the experimental conditions em-
ployed, rpoH expression in JF9 reached maximal levels, and in-
duction above the observed level was not possible or detectable.
MtrR directly binds to the rpoH promoter region. Initial
EMSA experiments showed that MtrR bound to the DNA
sequence upstream of rpoH encompassing its promoter region
in a specific, concentration-dependent manner and did not
FIG. 4. MtrR regulation of the RpoH-regulated grpE gene. Shown are the ?-Gal-specific activities expressed in grpE-lacZ fusion strains JF11
(MtrR positive) and JF12 (MtrR negative) and the mtrR?complemented strain JF13. The results are shown as average values (? standard
deviations) from three independent assays. The differences between strains JF11 and JF12 and between JF12 and JF13 were significant (P ? 0.001
and 0.003, respectively).
FIG. 5. H2O2induction of rpoH expression. Shown in the graph are the ?-Gal-specific activities in rpoH-lacZ fusion strain JF8 after a 15-min
exposure to 1 mM H2O2.The results are shown as average values (? standard deviations) from three independent assays; the difference was
significant (P ? 0.00001). The inset shows the changes in expression for mtrR, rpoH, and grpE when fusion strains were exposed to H2O2.
VOL. 191, 2009MtrR REGULATION OF rpoH293
require the 172-bp leader sequence (data not presented). In
order to determine if MtrR directly or indirectly regulates the
RpoH-regulated grpE gene, an EMSA was performed using
MtrR and a 305-bp probe containing the upstream DNA (in-
cluding a putative promoter element) of the grpE coding se-
quence. Unlike its binding to the DNA sequence upstream of
mtrC and rpoH, MtrR failed to bind the grpE target DNA
sequence (data not presented). Accordingly, we believe that
the ability of MtrR to regulate grpE is indirect and due to its
capacity to repress rpoH.
In order to identify the MtrR-binding site within the rpoH
promoter region, a DNase I protection assay was performed.
The rpoH promoter probe described above was radiolabeled
separately at its 5? and 3? ends and utilized in DNase I pro-
tection assays that employed increasing amounts (0 to 20 ?g)
of MtrR (Fig. 6). With increasing amounts of MtrR, an area of
protection of seventeen nucleotides on the sense strand (Fig.
2) could be identified as being involved in binding MtrR. A
second area of protection of sixteen nucleotides was observed
on the antisense strand (Fig. 2), which overlapped the area
observed on the sense strand. The region of protection indi-
cates that MtrR binds to the rpoH promoter at an area that
overlaps the ?35 hexamer sequence of the rpoH promoter.
Using the nucleotide sequence of the MtrR-binding site on the
noncoding strand within the mtrCDE promoter (5?-GGCACG
TTAGCACATA-3?) defined by Hoffman et al. (12), we were
able to identify a region of similarity (73.3% identity) (Fig. 2)
within the rpoH promoter region, which was also found by
DNase I protection to bind MtrR.
Modulation of MtrR expression and levels of antimicrobial
resistance. In order to further connect MtrR expression with
levels of rpoH expression and to assess the biologic conse-
quence of MtrR gene control in gonococci, we next inserted an
IPTG-inducible copy of the wild-type mtrR gene from strain
FA19 into its transformant strain JF9 (FA19 ?mtrR rpoH-lacZ)
to create strain JF7 (FA19 ?mtrR mtrR?rpoH-lacZ). Strain
JF7 was grown in GCB broth to mid-log phase and split into
two samples, one receiving 1 mM IPTG and one left un-
treated. After 1 h of induction, MtrR was clearly produced
by the IPTG-treated culture (Fig. 7B), and rpoH expression
was decreased by nearly twofold (Fig. 7C) compared to the
untreated sample. In order to determine whether induction
of mtrR expression resulted in biological changes of gono-
cocci, we next examined the susceptibility of the IPTG-
treated and control cultures to Ery and H2O2. Ery suscep-
tibility analysis was performed because MtrR repression of
the mtrCDE-encoded efflux pump increases gonococcal sus-
ceptibility to this antibiotic and other hydrophobic antimi-
crobial agents (10, 11, 19). In support of this, we found that
inducible expression of mtrR resulted in increased suscepti-
bility of gonococci to Ery (Fig. 7D). H2O2assays were per-
formed because loss of RpoH production was found to en-
hance the susceptibility of Brucella melitensis (1) to the
oxidizing agent. Since we (unpublished observations) and
others (3, 8, 14) have been unsuccessful in obtaining an rpoH
null mutant, we hypothesized that under conditions where
rpoH expression is dampened due to the repressive action of
MtrR, gonococcal susceptibility to H2O2would increase.
Indeed, we found that inducible expression of mtrR in-
creased gonococcal susceptibility to H2O2(Fig. 7E).
We believe that the MtrR regulon defined in this study
represents the minimum number of gonococcal genes under
MtrR control, as other genes (farR, ponA, and pilMNOPQ)
known (6, 15) to be controlled by MtrR were not identified as
being regulated by ?1.5-fold in the microarray data set. In this
respect, the MtrR-repressed farR (15) and pilM (6) genes were
measured as being repressed by 1.44- and 1.40-fold, respec-
tively, in the microarray data set, while the MtrA-activated
ponA gene was measured to be 1.24-fold activated. Other
MtrR-controlled genes might be uncovered under different
growth conditions or by exposure of gonococci to stress con-
ditions or antimicrobial agents.
Although the microarray results indicated that expression of
rpoH was modestly repressed by MtrR, the level of repression
(1.58-fold) was only slightly less than those of the mtrCDE
genes (1.7- to 2.2-fold). Since loss of MtrR repression of
mtrCDE in strain JF1 (FA19 ?mtrR) can have important con-
sequences for certain gonococcal properties (levels of antimi-
crobial susceptibility and in vivo fitness ), we elected to use
rpoH as a model gene outside of the mtrCDE locus to study the
FIG. 6. Identification of the MtrR-binding site within the rpoH
promoter. The MtrR-binding site (see the nucleotide sequences in Fig.
2 and its legend) was identified by DNase I protection using increasing
amounts of MtrR (0, 5, 10, and 20 ?g) in assays that employed sense
and antisense probes. The site of protection on each strand is shown by
the vertical bar adjacent to the protected region. The nucleotide-
sequencing reaction for each strand is shown (G, A, T, C) adjacent to
the lanes with the protection reactions.
294 FOLSTER ET AL.J. BACTERIOL.
global regulatory properties of MtrR. The combined transla-
tional fusion data that examined rpoH expression in the
presence or absence of MtrR, especially those involving the
inducible production of MtrR (Fig. 7), the increased expres-
sion of three RpoH-activated genes (clpB, marR, and grpE)
in the MtrR-deficient strain JF1, and the ability of MtrR to
bind within the rpoH promoter (Fig. 6), led us to conclude
that MtrR acts as a direct repressor of rpoH. As EMSA
experiments failed to show MtrR binding to grpE, we also
conclude that the ability of MtrR to control the expression
of this RpoH-regulated gene is likely indirect and is most
easily explained by the ability of MtrR to dampen rpoH
What is the biological significance of MtrR regulation of
rpoH? We considered the possibility that because rpoH expres-
sion can enhance bacterial resistance to killing by H2O2(1) and
because certain genes under RpoH control are upregulated in
gonococci during bacterial exposure to H2O2(29), MtrR ex-
pression in gonococci would impact the levels of such resis-
tance. The results presented here support this hypothesis.
Moreover, based on these results, we suggest that MtrR is a
general regulator of levels of gonococcal susceptibility to an-
timicrobial agents of the innate host defense, particularly the
nonoxidative and oxidative killing systems of neutrophils.
Thus, by repressing mtrCDE expression, MtrR can enhance
gonococcal susceptibility to LL-37, which is produced by neu-
trophils, epithelial cells, and certain organs (e.g., testis) (24)
and is a mediator of nonoxidative killing of bacteria. As MtrR
production also increases bacterial susceptibility to H2O2, its
control of gene expression, along with other resistance systems,
may be important in the ability of gonococci to survive oxida-
tive killing by neutrophils. We do not yet know if the observed
decrease in H2O2susceptibility in mtrR mutants directly or
indirectly involves RpoH, but the ability of MtrR to control
rpoH expression suggests that this regulatory scheme is impor-
tant in the general stress response. We also noted that hsp33
FIG. 7. Inducible production of MtrR represses rpoH expression and modulates antimicrobial susceptibility levels in gonococci. (A and B)
Strain JF7 was grown in the presence or absence of 1 mM IPTG as described in Materials and Methods, and cell lysates before induction (0 Hour)
or after 1 h of incubation in the absence (?) or presence (?) of IPTG were solubilized and subjected to SDS-PAGE using a 12.5% (wt/vol)
SDS-PAGE gel with the separated proteins stained by Coomassie brilliant blue (A); the positions of the molecular mass markers are shown on
the left of the gel, and the approximate positions of MtrR as determined by immunoblotting with detection using anti-MtrR antiserum (B) are
shown. (C) Specific activity values for ?-Gal levels in the control (?IPTG) and induced (?IPTG) cultures. The results are shown as average values
(? standard deviations) from three independent assays. The difference was significant (P ? 0.0012). (D) The efficiencies of plating of the control
and IPTG-induced cultures on GCB agar with or without 0.5 ?g/ml of Ery are shown. (E) The H2O2susceptibilities of the control and
IPTG-induced cultures were assessed by a disk diffusion assay, and the difference in growth inhibition between the cultures was significant (P ?
VOL. 191, 2009 MtrR REGULATION OF rpoH295
expression is also subject to MtrR repression (2.24-fold) (Ta-
ble 3). This is of interest because Hsp33 has been implicated in
bacterial responses to heat and peroxide (32). Unlike, grpE,
MtrR regulation of hsp33 may be direct, as a potential MtrR-
binding site consisting of 31 bp is positioned from nucleotides
?110 to ?141 (data not presented); this putative binding site
is 61.3% identical to the 31-bp region upstream of mtrCDE
shown by Lucas et al. (16) to bind MtrR. Taking these data
together, we propose that naturally occurring mtrR mutants
(22, 23, 34, 35) may have an advantage during infection, based
on their increased resistance to LL-37 (24) and H2O2(Fig. 7).
This linkage of bacterial resistance to distinct mediators of
innate host defense through MtrR may help to explain the
recent observation of Warner et al. (31) that mtrR mutants
have a competitive advantage over otherwise wild-type gono-
cocci in a female mouse lower genital tract infection model.
The potential roles of other transcriptional regulators similar
to MtrR in modulating bacterial resistance to innate host de-
fense mechanisms should be considered.
Against this background, it should be asked why mtrR mu-
tants are not the dominant strain in the community. At first
glance, it seems to be a disadvantage for gonococci to have
maintained mtrR, as its presence would downregulate the ex-
pression of genes needed for resistance to antimicrobials. We
propose that because MtrR can also transcriptionally activate
certain genes (Table 3) involved in metabolism (e.g., abpE,
glnE, and rfbB), the need to resist host-derived antimicrobials
through constitutive MtrR-repressible systems may be essen-
tial only at those sites rich in these agents. For most strains and
infections, the need to have expression of MtrR-activated
genes is more critical and overrides any advantage afforded by
mtrR mutations, particularly since the mtrCDE efflux operon
can be transcriptionally induced by MtrA in the presence of
sublethal levels of antimicrobials (20), even in the presence of
a functional MtrR.
We thank L. Pucko for help in manuscript preparation and submis-
sion, N. Kamal and Y. Zalucki for critical reading of the paper, M.
Apicella for facilitating access to the microarray slides, and the staff at
the University of Iowa DNA Facility for sample preparation and hy-
This work was supported by NIH grants AI-21150 (W.M.S.), RR-
015564 (J. Iandolo and D.W.D.), and RR016478 (F. Waxman and
D.W.D.) and by a VA Merit Award to W.M.S. W.M.S. was supported
by a Senior Research Career Scientist Award from the Department of
Veterans Affairs Medical Research Service.
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