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The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site ('marbox') at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thrl2Ala, Arg36Ala, Thr95Ile, and Prol06Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA. doi: 10.1128/JB.00103-10

Cationic antirnicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human [beta]-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human [beta]-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human [alpha]-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs. DOI 10.1099/mic.0.033415-0

Bacterial two-hybrid studies of randomly cloned Escherichia coli DNA identified a physical interaction between GyrA, subunit A of gyrase, and MarR, a repressor of the marRAB operon. GyrA-His immobilized on Ni-nitrilotriacetic acid (NiNTA) resin bound MarR, while MarR alone did not bind. GyrA interfered with MarR binding to marO, as detected by electrophoretic mobility assays. In a strain bearing the marRAB operon and a marO-lacZ reporter, overexpression of GyrA increased LacZ activity, indicating decreased repression of marO-lacZ by MarR. These results were confirmed by an increased survival of cells treated with quinolones and other antibiotics when GyrA was overexpressed. This work, like a previous study examining TktA (12), shows that unrelated proteins can regulate MarR activity. The findings reveal an unexpected regulatory function of GyrA in antibiotic resistance. doi: 10.1128/JB.01259-09

MarA, a transcriptional regulator in Escherichia coli, affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of hdeAB and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of hdeAB by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of hdeAB. Moreover, repression of hdeAB by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of hdeAB by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on hdeAB under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of hdeAB expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of hdeAB by MarA depends on pH, growth phase, and other regulators of hdeAB and is associated with reduced resistance to acid conditions.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.03.004 Byline: Michael N. Alekshun (1), Stuart B. Levy (2) Abstract: Treatment of infections is compromised worldwide by the emergence of bacteria that are resistant to multiple antibiotics. Although classically attributed to chromosomal mutations, resistance is most commonly associated with extrachromosomal elements acquired from other bacteria in the environment. These include different types of mobile DNA segments, such as plasmids, transposons, and integrons. However, intrinsic mechanisms not commonly specified by mobile elements -- such as efflux pumps that expel multiple kinds of antibiotics -- are now recognized as major contributors to multidrug resistance in bacteria. Once established, multidrug-resistant organisms persist and spread worldwide, causing clinical failures in the treatment of infections and public health crises. Author Affiliation: (1) Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA (2) Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology & Microbiology and Department of Medicine, Tufts University School of Medicine, Boston, MA 02111, USA

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coil isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo. DOI: 10.1099/mic.0.2006/000604-0

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of Ion (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.

We examined whether household use of antibacterial cleaning and hygiene products is an emerging risk factor for carriage of antimicrobial drug-resistant bacteria on hands of household members. Households (N = [...]

Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330-20339]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short [alpha]-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

Collaboration between networks presents opportunities to increase analytical power and cross-validate findings. Multivariate analyses of 2 large, international datasets (MYSTIC and SENTRY) from the Global Advisory on Antibiotic Resistance Data [...]

Studies were undertaken to determine the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.

Research has been conducted on the TetA efflux pump protein. The study of the site where tetracyclin binds to this pump protein has been carried out and the results indicate that when chelated to tetracyclin Fe (super)2+ cleaves TetA backbone at glutamine 225 in transmembrane helix 7.

The Pseudomonas fluorescens transcription activator AdnA has been found to be necessary for motility and adhesion. Both are in a gene showing homology to the NtrC/NifA family of transcription activators. The phenotypes seen are caused by lack of AdnA and are not the result of other mutation or polar effects of mutations in adnA on other genes. The insertions Pf0-5 and Pf0-10 have been localized. The disrupted genes are now known to be the same ORF. Recently found data indicate that in P. aeruginosa, the FliD protein, at the tips of flagella, mediates a specific interaction with mucins.

The transcription factor AdnA of Pseudomonas fluorescens allows the organism to spread and survive in soil. This is important because this organism is considered to be beneficial in treating plant diseases, decontaminating soil, and improving plant nutrition.

Research has been conducted on the MarA proteins in Escherichia coli. Results indicate that the mar locus mediates a global stress response that involves chromosomal genes.

The multiple antibiotic resistance locus (mar) of Escherichia coli regulates intrinsic susceptibility to multiple antibiotics, organic solvents, oxidative stress agents and household disinfectants. MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. Research was conducted which revealed that sodium salicylate, plumbagin, 2,4-dinitrophenol and menadione-inducers of the marRAB operon in whole cells, interfered with the repressor activity of Mar R in vitro.

The existence of mar-like regulatory drug resistance response was investigated in Mycobacterium smegmatis by analyzing antimicrobial vulnerability in cells that express the cloned Escherichia coli marA gene. The expression resulted in increased resistance to multiple antimicrobial agents, such as chloramphenicol, tetracycline, ethambutol, isoniazid and rifampicin. Changes in resistance did not materialize when the plasmid bearing marA was lost.

A study using site-directed mutagenesis was conducted to examine the role of glutamate residues located within putative transmembrane helices in TetA(P)-mediated tetracycline efflux. The results indicate that the negatively charged residues that seem to be located within or near the membrane play an essential role in the function of TetA(P). Moreover, different helices in different Tet proteins may have homologous functions.

A research study was conducted regarding the role of acr and multiple antibiotic resistance (mar) loci in organic solvent tolerance mediated by expression of marA, soxS or robA in Escherichia coli (E coli). The methods used were the DNA manipulations and organic solvent tolerance analysis. Results showed that marA, soxS and robA can independently create organic solvent tolerance in E coli with the presence of acrAB locus.

Transcriptional fusion of marOII-lacZ into the chromosome of wild-type and purified marR mutants was examined to determine the factors affecting the expression of the marRAB operon in Escherichia coli. Wild-type cells exhibited a two- to seven-fold increase in LacZ expression, while mar-deleted cells exhibited a 20-fold increase. Results of the fusion of MarR with a fusion protein MarE indicated repressor ability and a dissociation constant of 5 x 10(sub -9) M, suggesting that repression in cells are reversed to facilitate the response of the bacteria to stress.

A study of the participation of non-marRAB chromosomal loci in chromosomal multiple antibiotic resistance (Mar) phenotype using TrophoA and TnlacZ elements helps study mar-sensitive fusions in a specially designed Escherichia coli recipient that has a temperature-sensitive plasmid carrying the mar locus and the mar RAB operon. Five mar-regulated gene fusions are detected, with beta-galactosidase expression being stimulated in two of them and alkaline phosphate expression being suppressed in the other three. Occurrence of these changes due to marA alone indicates that MarA is the regulatory protein that leads to enhanced resistance to multiple antibiotics.

Two adhesion-defective mutants, Pf0-10 and Pf0-15, with different Tn5 insertion positions, are isolated from Pseudomonas fluoresences. Pf0-15 contains high concentrations of a 34-kDa outer membrane protein and flagella for mobility, unlike Pf0-10 which lacks both features. The mutants exhibit discrepancies in initial attachment steps but reveal a slow final binding phase.

Three pleiotropic mutations of the 34 min mar locus of Escherichia coli activate the marRAB operon that probably triggers antibiotic resistance and oxidative stress genes by relieving the repressive effects of MarR protein. the pleiotropic effects are reversed to varying degrees by marR+ gene or a multicopy plasmid. The underlying mechanisms of these marR1, soxQ1 and cfxB1 mutants are possibly similar as shown by studies of RNA expression.

An analysis of the influence of salicylate on the expressin of the marRAB operon reveals that salicylate activates expression of the marRAB operon, which indicates involvement of the mar locus in phenotypic antibiotic resistance (PAR). Development in salicylate in both mar-lacking and mar-containing strains leads to enhanced antibiotic OmpF, enhanced micF transcription and reduced permeation of the external membrane to cephaloridine. However, these changes occur more in cells which contain mar.

Escherichia coli multiple antibiotic resistance locus from a 7,800 base-pair region in the chromosomal DNA has been sequenced. A sequence within this region has the potential to be a divergent promoter which includes a segment with two pairs ofdirect repeats, marO. Flanking marO are putative open reading frames (ORF) three on either side. Mutations in the downstream ORFs were characterized.

A Tn5 insertion which led to increased susceptibility to multiple drugs, including tetracycline, chloramphenicol, nalidixic acid, erythromycin, spectinomycin, norfloxacin and novobiocin, was identified in Escherichia coli. Cloning and sequence analysis showed that this locus is identical to prc gene, which map at min 40.4. The prc product has beenlinked to proteolytic processing of penicillin-binding protein 3. Moreover, theresults indicate that the prc gene product affects sensitivity to multiple antibiotics possibly by influencing cell permeability.

The role of the aspartate 15 residue of the class B tetracycline efflux protein Tet was studied, using site-directed mutagenesis in Escherichia coli. Replacement of aspartate by tyrosine, histidine or asparagine resulted in a reduction in tetracycline resistance. The tyrosine and histidine substitutions lowered the Vmax of tetracycline transport but did not change the Km. On the other hand, the asparagine substitution increased the Km but did not change the Vmax. Therefore, Asp-15 could play a role in Tet efflux.