The Escherichia coli mar Locus—
Antibiotic Resistance and More
The mar locus and related systems confer multiple antibiotic
resistance and control expression of virulence factors and genes for
metabolizing small molecules
Michael N. Alekshun and Stuart B. Levy
he multiple antibiotic resistance
(mar) locus of Escherichia coli was
first described in the early 1980s during efforts to characterize the genetic
basis of tetracycline-resistant mutants derived in vitro. Originally selected on subinhibitory concentrations of tetracycline, these
mutants also exhibited cross-resistance to other
antibiotics, including fluoroquinolones and ␤-lactams. The Mar phenotype is inducible by salicylate, salicylate-like compounds, and aryloxoalcanoic acids. Inducible Mar and Mar-like systems
are found in Yersinia spp., Salmonella enterica
serovars Choleraesuis, Typhimurium, and Paratyphi B, Enterobacter aerogenes, Pseudomonas
aeruginosa, Burkholderia cepacia, Mycobacterium tuberculosis, and Staphylococcus aureus.
The Mar phenotype in E. coli depends on the
activity of two transcription factors, MarR (multiple antibiotic resistance repressor) and MarA
(multiple antibiotic resistance activator), which
are members of the helix-turn-helix (HTH) superfamily of proteins. MarR is a representative of the
winged-helix subfamily, and MarA is a member of
the AraC subfamily. There are numerous MarR
and AraC orthologs/paralogs that are widely distributed among both the Bacteria and the Archaea, among which they play roles in regulating
multiple and single antibacterial resistances, microbial virulence, and bacterial physiology.
T
MarB (Fig. 1). Expression of marRAB is regulated by the activity of MarR and MarA. When
MarR is active, expression is repressed; when it
is inactivated by small molecules or mutated,
marRAB transcription increases. Of prokaryotic and eukaryotic genomes examined, more
than 1,400 AraC family members were found
among prokaryotes and only isolated representatives were reported among eukaryotes (Fig. 2).
Whether the latter represent true genome members or prokaryotic contaminants is unknown.
AraC proteins can be divided into two general
classes. One class includes small (⬃15-kDa)
proteins such as E. coli MarA and SoxS that
contain only a DNA-binding domain (DBD).
Other AraC-like proteins, such as E. coli Rob
and AraC and P. aeruginosa ExsA, are much
larger (⬃30 kDa) and contain both a DBD and
another domain of either known or unknown
function.
The marC gene, which specifies a 221-aminoacid (aa) inner membrane protein of unknown
function, is well conserved in a number of bacterial genera. marB codes for a small putative
protein (72 aa) also of unknown function; the
gene has so far been found only in E. coli,
Shigella flexneri 2a, and S. enterica serovar Typhimurium (S. typhimurium).
Regulons: a Panoply of Genes Regulated
by MarA and other AraC Family Members
The mar Locus: Its Components
and Regulation
The E. coli marCRAB locus consists of four
genes specifying MarC, MarR, MarA, and
MarA, SoxS, and Rob act as pleiotropic transcription factors. Each protein directly regulates
the expression of multiple genes, termed the
MarA, SoxS, and Rob regulons (Fig. 3A). These
Michael N. Alekshun is Associate
Director of Antiinfective Discovery,
Paratek Pharmaceuticals, and Stuart B.
Levy is Professor of
Molecular Biology
and Microbiology
and of Medicine,
Director of the Center for Adaptation
Genetics and Drug
Resistance, Tufts
University School of
Medicine; Chief
Scientific Officer,
Paratek Pharmaceuticals
Volume 70, Number 10, 2004 / ASM News Y 451
Baltimore County, Baltimore,
Md., and Robert Martin and Lee
Rosner at the National Institute of
Diabetes and Digestive and Kidney
Diseases in Bethesda, Md. Moreover, MarA can also act directly as
a repressor, and this function is
apparently affected by the position
of the MarA binding site (the marbox), according to our collaborator Thamarai Schneiders. Similarly Demple and his group report
that Rob has repressor activity.
Martin and Rosner recently
Genetic organization of the E. coli mar locus. The marRAB operon is negatively (⫺) regulated
by MarR (multiple antibiotic resistance repressor) and positively (⫹) regulated by MarA
used both standard molecular bi(multiple antibiotic resistance activator and global transcription regulator) via their binding to
ology techniques and bioinformatthe mar promoter/operator region. The functions of MarB and MarC are unknown.
ics to cross-validate a number of
the genes identified in the MarA
and SoxS regulons. Their data suggest that migene products are responsible for protecting the
croarray experiments tend to overestimate the
cell from a variety of stress conditions. Because
numbers of genes within these regulons, thus
of redundancy among these regulons, the cell is
lowering the estimate for the combined MarA/
well poised to respond to toxic insults such as
SoxS/Rob regulon to about 50 genes.
antibiotics, even in the absence of any one transcription factor.
Researchers using genomic arrays, molecular
Clinical and Environmental
genetics, bioinformatics, and other methods to
Relevance of mar
characterize the E. coli MarA, SoxS, and Rob
Some clinical isolates of E. coli, Enterobacter
and P. aeruginosa ExsA regulons have reached
cloacae, Klebsiella pneumoniae, and S. typhisimilar, but also different conclusions about
murium constitutively express MarA or a rehow these regulons work.
lated transcription factor. Several research
For instance, in a strain of E. coli with a
groups use the organic solvent tolerance (OST)
plasmid constitutively expressing marA this
phenotype exhibited by Mar mutants to identify
regulon involved expression of 62 genes, acclinical strains that overexpress MarA or a
cording to our former colleague Teresa Barbosa.
MarA ortholog.
Taking two different approaches, Bruce Demple
In a survey of 138 fluoroquinolone-susceptiand his collaborators at the Harvard School of
ble (FQS) and -resistant (FQR) clinical isolates,
Public Health, in Boston, Mass., analyzed Robwe found that about 2% of the FQS but 30% of
responsive genes and also characterized the
the FQR isolates were cyclohexane tolerant;
MarA and SoxS regulons as well as the cell’s
40% of the latter isolates constitutively exresponse to sodium salicylate (a MarR antagopressed either marA or soxS. Among 19 FQR
nist and inducer of marRAB expression) and
clinical Klebsiella isolates, 52% were tolerant to
paraquat (an inducer of the soxRS system). Deorganic solvents, while about 16% of those
spite identifying differences, they also concluded
strains overexpressed RamA, a MarA ortholog.
that the number of genes whose expression inAccording to Robert Tibbetts and colleagues
creases outnumbered those with reduced exat Purdue University in West Lafayette, Ind.,
pression by a ratio of 3:1 in response to MarA.
about 34% of S. enterica serovar Choleraesuis
In contrast, SoxS down-regulates approximately
isolates (n ⫽ 53) from pigs were OST and multhree times as many genes as it up-regulates. We
tiple-drug resistant (MDR); several overexalso find several intergenic regions are responpressed marA. Similarly, S. typhimurium exsive to MarA.
pressed resistance to an E. coli-secreted
microcin following exposure to salicylate, and
MarA directly activates genes, according to
Richard Wolf, Jr., at the University of Maryland
MarR modulated this phenotype, according to
FIGURE 1
452 Y ASM News / Volume 70, Number 10, 2004
Steve Carlson and his collaborators at
the U.S. Department of Agriculture Agricultural Research Service, Ames,
Iowa. Approximately 10% of 388 Salmonella serotypes of animal origin
were OST and bore a MDR phenotype
that included resistance to antibiotics
and disinfectants such as triclosan and
cetrimide, according to Martin Woodward and his collaborators at Veterinary Laboratories Agency in Weybridge, Surrey, United Kingdom.
Resistance to household disinfectants
is associated with MarA and SoxS overexpression. Because so many antibiotics
used in the United States are also administered for purposes of animal husbandry, isolates of animal as well as
human origin could serve as reservoirs
of MDR strains.
FIGURE 2
Distribution of AraC proteins among representative genomes (taken from the InterPro
database [http://www.ebi.ac.uk/interpro/]). Organisms in which AraC proteins are found
are indicated in boldface; the notation in parenthesis represents the number of
occurrences of proteins within the host(s). AraC proteins have not been reported in those
organisms labeled in grey.
lates, Hiroshi Nikaido at the University of California, Berkeley, and his collaborators found that
MarA-mediated resistance depends on increased exMany AraC family members play roles in antipression of AcrAB, a multidrug efflux pump. AcrAB
biotic resistance and microbial virulence (Table
likely protects against biliary salts, an obvious ad1). For example, overexpression of MarA in E.
vantage to a microbe passing through the digestive
coli produces drug resistance and attenuates the
tract of a mammalian host.
rapid bactericidal activity of fluoroquinolones.
Nonionic detergents, such as triton X-100 and
MarA-mediated drug resistance in these and
nonoxynol-9, can induce the MtrCDE efflux system
other organisms is in large part achieved
of Neisseria gonorrhoeae, and this inducibility is
through increased expression of a multidrug
dependent on MtrA (another AraC protein), accordefflux system. While a correlation between drug
ing to William Shafer of Emory University School of
efflux and resistance was first suggested in studMedicine, Atlanta, Ga. He and his colleagues find
ies involving both susceptible and resistant isothat MtrCDE protects pathogens from antimicrobial
agents present on mucosal
surfaces of infected mice.
The dramatic effects on
Deletion of AraC family members attenuates pathogenesis in murine
pathogenesis (Table 1) folmodels of infection.
lowing the deletion of a gene
Organism
LD50 (CFU)
Inoculum (CFU)
# of mice dead at
specifying an AraC-like pro36 hr
168 hr
tein is in part attributed to
the ability of these proteins
V. cholerae
to coordinate the expression
O395, wta
1.5 ⫻ 1010
8/8
VJ740, ⌬toxT
7 ⫻ 109
2/7
of large numbers of genes in
P. aeruginosa
“virulence regulons.” The
PA103, wt
5 ⫻ 107
5/5
genes
within these regulons
7
PA103, ⌬exsA
5 ⫻ 10
1/5
specify
proteins that reguY. pestis
late the expression of memKIM-53, wt
1
⌬lcrF
⬎100
brane proteins involved in
MDR, type III secretion sysa
wt, wild type.
tems, microbial toxins (e.g.,
cholera toxin), biofilm forMarA Paralogs and Orthologs: Roles in
Antibiotic Resistance and Virulence
Volume 70, Number 10, 2004 / ASM News Y 453
FIGURE 3
Francisco and Victor DiRita at the University of Michigan Medical School in
Ann Arbor.
In a mouse model of pyelonephritis,
we recently found that E. coli lacking
MarA, SoxS, and Rob cannot colonize
kidneys. Also, Proteus mirabilis UreR
(an AraC protein) is required to infect
the urinary tracts of mice, according to
Harry Mobley at the University of
Maryland School of Medicine, Baltimore, Md. When tested in chickens, S.
typhimurium DT104 strains lacking
marA are less frequently isolated from
the spleens and caecal contents of infected chicks than are parent strains
containing marA, according to Martin
Woodward and his colleagues in the
United Kingdom.
Three-Dimensional Structures
of MarA and Rob
The three-dimensional structures for
both the E. coli MarA and Rob proteins
in complex with their DNA substrates
are now known (Fig. 4). These structural data directly support a wealth of
genetic and biochemical evidence that
first suggested the presence of the
unique dual helix-turn-helix (HTH)
DNA binding motif that defines this
protein family. Surprisingly, this motif
The E. coli MarA regulon and the proposed function of a small-molecule transcription
also occurs in the ␭ integrase, a protein
factor inhibitor. (A) MarA (or other AraC family member) in its active state functions freely
as a transcription factor to regulate (“lights-on”) a virulence regulon. (B) The presence of
that is not a transcription factor.
a small-molecule inhibitor would prevent (“lights-off”) the expression of the regulon.
In the crystal structures, MarA enTranscription regulation may be indirect, direct, positive, or negative. MDR, multiple drug
gages its target with both N- and Cresistance.
terminal HTH motifs, thus bending the
DNA (Fig. 4A), whereas Rob shows
mation, carbohydrate transport and metaboonly a N-terminal HTH in its interaction with a
linear DNA fragment (Fig. 4B). Although these
lism, cell envelope synthesis, and lipid metabotwo crystal forms differ, each is supported by
lism (Fig. 3A).
either additional biochemical data or further
Deleting the gene specifying LcrF in Yersinia
biophysical characterizations.
pestis KIM53 results in a substantial decrease in
the infectivity of this organism (Table 1), according to Yehuda Flashner and colleagues at
AraC Family Members as Targets of New
the Israel Institute for Biological Research, Ness
Anti-infection Therapeutics
Ziona, Israel. Similarly, removing either exsA in
Antibiotic resistance represents a growing pubP. aeruginosa or toxT in Vibrio cholerae renders
lic health problem. For instance, methicillineach of these organisms virtually avirulent in
resistant Staphylococcus aureus (MRSA) and
mice (Table 1), according, respectively, to
Joanne Engel at the University of California, San
multidrug-resistant uropathogenic E. coli are
454 Y ASM News / Volume 70, Number 10, 2004
now widespread in communities. Some
FIGURE 4
clinical isolates of MRSA also contain
high-level resistance to vancomycin,
which is considered a “drug of last resort.”
Although the fluoroquinolones are
the mainstay for treating common urinary tract infections, resistance to these
agents among gram-negative bacilli is
climbing in parallel to the increased use
of these drugs. Several large pharmaceutical companies have stopped their
antibiotic research and development
programs, in part because of economic
concerns, leaving the burden of drug
development to smaller companies.
A novel approach for preventing or
treating infections without risking deComparison of the E. coli MarA (A) and Rob (B) co-crystal structures.
velopment of drug resistance is needed
to meet the threat of future MDR infectious diseases. For instance, Michael
ily proteins from E. coli, S. typhimurium, P. miraGivskov and colleagues at the Technical Univerbilis, and P. aeruginosa. These novel agents inhibit
sity of Denmark, Lyngby, Denmark, recently
the DNA binding activity in vitro of multiple AraC
identified small molecules that inhibit quorum
transcription factors. Of note, these agents prosensing in P. aeruginosa, and these compounds
duce the attenuated virulence phenotype exhibited
exhibit activity in vivo.
by mutant E. coli in the murine pyelonephritis
The targeting of a family of bacterial tranmodel.
scription factors like AraC family members for
The therapeutic potential of transcription facanti-infection chemotherapy, however, would
tor
modulators is validated in cancer chemorepresent a unique opportunity. While most pretherapy,
atherosclerosis, inflammation, diabevious attempts to curtail bacterial virulence fotes,
Parkinson’s
disease, and in rheumatic and
cused on single virulence factors, including miother autoimmune diseases. Examples of clinicrobial adhesins such as pili and MSCRAMMs
cally effective products from these efforts into prevent adherence of the pathogen, the tarclude small molecules that target peroxisome
geting of a regulatory protein offers the advanproliferator-activated receptors, Hedgehog, and
tage of simultaneously affecting expression of a
liver X receptor. The potential of this technolnumber of virulence factors (Fig. 3B).
ogy in anti-infective therapy remains to be furWe recently identified groups of low-molecularther explored.
weight organic compounds that target AraC famACKNOWLEDGMENTS
The authors would like to thank Robert D. Arbeit and S. Ken Tanaka for their critical reading of the manuscript. We are also
indebted to Bonnie Marshall for her lead role in the production of Fig. 1 and 3. Due to space constraints we apologize for not
being able to cite all of the references for the work presented herein.
SUGGESTED READING
Alekshun, M. N., and S. B. Levy. 1999. The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends
Microbiol. 7:410 – 413.
Alekshun, M. N., and S. B. Levy. 1997. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon
Antimicrob. Agents Chemother. 41:2067–2075.
Barbosa, T. M., and S. B. Levy. 2000. Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive
expression of MarA J. Bacteriol. 182:3467–3474.
Dangi, B., P. Pelupessey, R. G. Martin, J. L. Rosner, J. M. Louis, and A. M. Gronenborn. 2001. Structure and dynamics of
MarA-DNA complexes: An NMR investigation. J. Mol. Biol. 313: 1067–1081.
Volume 70, Number 10, 2004 / ASM News Y 455
George, A. M., and S. B. Levy. 1983. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in
Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J. Bacteriol. 155:531–540.
Jair, K.-W., R. G. Martin, J. L. Rosner, N. Fujita, A. Ishihama, and R. E. Wolf, Jr. 1995. Purification and regulatory properties
of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters. J.
Bacteriol. 177:7100 –7104.
Levy, S. B., M. N. Alekshun, B. L. Podlogar, K. Ohemeng, A. K. Verma, T. Warchol, and B. Bhatia. Dec. 11, 2003.
Transcription factors modulating compounds and methods of use thereof. US Patent Application 2003/0229065.
Martin, R. G., and J. L. Rosner. 2003. Analysis of microarray data for the marA, soxS, and rob regulons of Escherichia coli.
Methods Enzymol. 370:278 –280.
Martin, R. G., and J. L. Rosner. 2001. The AraC transcriptional activators Curr. Opin. Microbiol. 4:132–137.
Pomposiello, P. J., M. H. Bennik, and B. Demple. 2001. Genome-wide transcriptional profiling of the Escherichia coli
responses to superoxide stress and sodium salicylate J. Bacteriol. 183:3890 –3902.
Schneiders, T., T. M. Barbosa, L. M. McMurry, and S. B. Levy. 2003. The Escherichia coli transcriptional regulator MarA
directly represses transcription of purA and hdeA. J. Biol. Chem. 279:9037–9042.
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