The MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognised. The signature of the family is amino acid similarity in the first 100 amino acids, including a helix-turn-helix motif followed by a coiled-coil region. With increasing recognition of members of this class over the last decade, particularly with the advent of rapid bacterial genome sequencing, MerR-like regulators have been found in a wide range of bacterial genera, but not yet in archaea or eukaryotes. The few MerR-like regulators that have been studied experimentally have been shown to activate suboptimal sigma(70)-dependent promoters, in which the spacing between the -35 and -10 elements recognised by the sigma factor is greater than the optimal 17+/-1 bp. Activation of transcription is through protein-dependent DNA distortion. The majority of regulators in the family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. A subgroup of the family activates transcription in response to metal ions. This subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region, but it is not yet clear how metal discrimination occurs. This subgroup of MerR family regulators includes MerR itself and may have evolved to generate a variety of specific metal-responsive regulators by fine-tuning the sites of metal recognition.

The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons.

The transcription factor FNR, the regulator of fumarate and nitrate reduction, regulates major changes as Escherichia coli adapts from aerobic to anaerobic growth. In an anaerobic glycerol/ trimethylamine N-oxide/fumarate medium, the fnr mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to reveal the response to oxygen, nitrate, and nitrite in the absence of glucose repression or artifacts because of variations in growth rate. Hence, many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved. The current microarray data confirmed 31 of the previously characterized FNR-regulated operons. Forty four operons not previously known to be included in the FNR regulon were activated by FNR, and a further 28 operons appeared to be repressed. For each of these operons, a match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 103, and possibly as many as 115, operons. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL, and a further 41 operons are repressed. The narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP ؉ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new defense mechanisms against reactive nitrogen species.In several recent studies, genome-wide transcription data have been analyzed to reveal the extent of the biochemical changes as Escherichia coli K12 adapts from aerobic to anaerobic growth. Salmon et al. (1) compared RNA isolated from cultures of strain MC4100 that had been grown aerobically or anaerobically and also from an anaerobic culture of an fnr mutant that lacks FNR, 2 the regulator of fumarate and nitrate reduction, which is a global regulator of many oxygen-regulated genes. In similar experiments, Kang et al. (2) grew both strain MG1655, for which the complete genome sequence is available, and an isogenic fnr mutant aerobically and anaerobically in a minimal salts medium, and compared their transcriptome data with those from the previous study. In both groups of experiments, glucose was used as the carbon source for growth, and in both studies rigorous statistical methods and cluster analysis were used to analyze the data. In the former study, expression levels of 1,445 genes changed in response to the availability of oxygen. Although the corresponding figure in the study of Kang et al. (2) was 962, only 334 genes were common to both data sets, and of those, 123 appeared to be regulated in opposite directions. Thus only 211 genes showed similar responses, 10% of the 2073 genes for which changes were observed.Both of the previous studies were valuable in revealing the far greater extent of chan...

We have shown that the open reading frame ybbI in the genomic sequence of Escherichia coli K‐12 encodes the regulator of expression of the copper‐exporting ATPase, CopA. In vivo studies showed that ybbI (designated cueR for copper export regulator gene) was required for copper tolerance during growth, that disruption of cueR caused loss of copA expression and that copA gene expression was regulated by cueR and by copper or silver ions. Expression of a lacZ reporter gene under the control of the copA promoter was approximately proportional to the concentration of cupric ions in the medium, but increased more rapidly in response to silver ion concentrations. The start of the copA transcript was located by primer extension mapping, and DNase I protection assays showed that the CueR protein binds in vitro to a dyad symmetrical sequence within a 19 bp spacer sequence in the copA promoter. CueR binding occurs in vitro in both the presence and the absence of RNA polymerase with or without copper ions present but, in the presence of CueR, RNA polymerase and copper ions, permanganate‐sensitive transcription complexes were formed. CueR is predicted to have an N‐terminal helix–turn–helix sequence and shows similarity to MerR family regulators.

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The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.The presence of toxic heavy metals in the environment has resulted in the development or acquisition by bacteria of genetic systems that counteract their effects. Many bacterial heavy metal resistance systems are based on efflux, and two groups of efflux systems have been identified. These can be either P-type ATPases, e.g., the Cu(II), Cd(II), and Zn(II) ATPases of gram-negative bacteria (31), or chemiosmotic pumps, e.g., the three-component divalent-cation efflux systems cnr, ncc, and czc of Ralstonia metallidurans (formerly Alcaligenes eutrophus) CH34 (35).Lead resistance has been reported in both gram-negative and gram-positive bacteria isolated from lead-contaminated soils, with Pseudomonas marginalis showing extracellular lead exclusion and Bacillus megaterium demonstrating intracellular cytoplasmic lead accumulation (28). Pb(II)-resistant strains of Staphylococcus aureus and Citrobacter freundii that accumulated the metal as an intracellular lead-phosphate have also been isolated (15, 16), though the molecular mechanism of detoxification remains to be elucidated. Efflux of Pb(II) has also been reported for the CadA ATPase of S. aureus and the ZntA ATPase of Escherichia coli (27).R. metallidurans strain CH34 contains at least seven determinants encoding resistances to toxic heavy metals, located on one of the two endogenous megaplasmids, pMOL28 and pMOL30 (for a review, see reference 35). One of these determinants, located on pMOL30 (20), mediates resistance to Pb(II). In this paper we describe the isolation and characterization of the Pb(II) resistance determinant, pbr, from pMOL30. MATERIALS AND METHODSBacterial strains, plasmids, and media. R. metallidurans and E. coli were grown in 869 medium (20) at 30 and 37°C, respectively. Antibiotic resistance was selected on media supplemented with 20 g of tetracycline or 100 g of ampicillin per ml, as appropriate. To test for Pb(II) resistance, cells were grown on RM medium, a modified 284 gluconate minimal medium (20) in which Tris-HCl is replaced by 20 mM morpholinepropanesulfonic acid (MOPS)-NaOH (pH 7) and Na...

Metals such as mercury, arsenic, copper and silver have been used in various forms as antimicrobials for thousands of years with until recently, little understanding of their mode of action. The discovery of antibiotics and new organic antimicrobial compounds during the twentieth century saw a general decline in the clinical use of antimicrobial metal compounds, with the exception of the rediscovery of the use of silver for burns treatments and niche uses for other metal compounds. Antibiotics and new antimicrobials were regarded as being safer for the patient and more effective than the metal-based compounds they supplanted. Bacterial metal ion resistances were first discovered in the second half of the twentieth century. The detailed mechanisms of resistance have now been characterized in a wide range of bacteria. As the use of antimicrobial metals is limited, it is legitimate to ask: are antimicrobial metal resistances in pathogenic and commensal bacteria important now? This review details the new, rediscovered and 'never went away' uses of antimicrobial metals; examines the prevalence and linkage of antimicrobial metal resistance genes to other antimicrobial resistance genes; and examines the evidence for horizontal transfer of these genes between bacteria. Finally, we discuss the possible implications of the widespread dissemination of these resistances on re-emergent uses of antimicrobial metals and how this could impact upon the antibiotic resistance problem. IntroductionMetals and metalloids have a long empirical history of human usage in medicine and agriculture (as reviewed below), despite problems of host toxicity or doubts about their efficacy. Even now, a few toxic metal(loid) compounds are still first-line drugs or preferred-choice chemotherapeutics or antimicrobials, although the use of most of the previously popular antimicrobial metal(loid)s, such as mercury and arsenic/antimony compounds, has been reduced or phased out in the past 50 or so years. Other metals, such as silver and copper, still have limited uses in agriculture and medicine, but are also increasingly being included in consumer products, from clothing to computer keyboards, and are being promoted as useful additions to our arsenal of antimicrobials. Against this background of their current usage, it is reasonable to ask: what is the relevance of antimicrobial metals and bacterial resistances to them to medical microbiology in the twentyfirst century?Any attempt to address this question must be set against the backdrop of widely known problems and opportunities. We are faced with new and emerging opportunistic nosocomial and community-acquired pathogens; and increasing epidemic and pandemic multidrug-resistant (MDR) pathogens. There is a recognition that the antibiotic discovery pipeline has not delivered significant quantities of new antibiotics in the past few decades, and new formulations and uses for antimicrobial metals as weapons in the antimicrobial armoury are being proposed (Department of Health, 2013;Lemire et al., 2013). ...

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