Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidus metallidurans CH34.

BACKGROUND: The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the -35 and -10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine. RESULTS: Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its -35 and -10 sites. Using ß-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the -10 sequence of PpbrA from TTAAAT to the consensus E. coli -10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the -10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using ß-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb(II)-induced activation of PpbrA. CONCLUSIONS: These data show that the metal-dependent activation of PbrR occurs by a similar mechanism to that of MerR, but that metal ion coordination is through cysteines which differ from those seen in other MerR family regulators, and that the DNA sequence of the -10 promoter affects expression levels of the lead resistance genes.

Transcriptional activation of MerR family promoters in Cupriavidus metallidurans CH34.

Metal responsive MerR family transcriptional regulators are widespread in bacteria and activate the transcription of genes involved in metal ion detoxification, efflux, or homeostasis, in response to the presence of cognate metal species in the cytoplasm. MerR family regulators recognize and bind to dyad symmetrical DNA sequences in specific promoters that have a spacer region between the -35 and -10 sequences which is longer than the canonical 16-18 bp spacer for other sigma(70)-dependent promoters. In this study we report beta-galactosidase assays of MerR family-regulated gene expression in the multiple metal resistant bacterium Cupriavidus metallidurans. A series of pMU2385 reporter plasmid derivatives containing cloned MerR family-activated promoters were used to determine metal ion-induced responses from different MerR family regulated promoters, as well as regulators cloned with the cognate promoter into pMU2385. Mercuric ion-responsive MerR and lead ion-responsive PbrR activity was confirmed using this assay system as well as MerR family activator activity on heterologous promoters PcopA, PcadA, and Pzcc from Escherichia coli, Pseudomonas aeruginosa and Bordetella pertussis, respectively. In C. metallidurans CH34, transcription from these promoters was activated by MerR family regulators encoded on the chromosome or megaplasmids in response to copper (PcopA), and lead (PcadA and PzccA), showing that MerR family activators in C. metallidurans can act on MerR family promoters from other organisms, which have sequence differences to the predicted C. metallidurans promoters.

Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter.

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg(2+) transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.

Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn501 mer operon.

Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg(2+)) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg(0)) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.

Zinc dependence of zinT (yodA) mutants and binding of zinc, cadmium and mercury by ZinT.

ZinT (B1973), previously known as YodA, was originally characterised as a cadmium-induced periplasmic protein under the regulation of Fur and SoxS. Here we describe a decrease in zinT transcript in response to elevated copper concentrations and the zinc and copper dependent phenotype of a DeltazinT strain. Cadmium sensitivity of the DeltazinT strain was not observed. We demonstrate the binding of nickel, zinc, cadmium, and mercury, but not cobalt, copper, iron, and manganese, to purified ZinT using mass spectrometry. This and previous studies support the hypothesis that ZinT plays a role in zinc homeostasis and is required for growth under zinc limited conditions, suggesting that ZinT is either a periplasmic zinc chaperone or is involved in zinc import. Limited metal ion discrimination results in regulation of PzinT in a non-specific manner, which is mirrored in the binding of several different heavy metals by ZinT.

The multicopper oxidase (CueO) and cell aggregation in Escherichia coli.

cueO encodes a periplasmic multicopper oxidase, which is known to be involved in copper homeostasis and protection against oxidative stress in Escherichia coli K12. Transcriptional profiling showed that expression of genes associated with motility was lowered in a cueO mutant while expression of genes associated with autoaggregation was elevated. Increased aggregation was correlated with increased expression of cell surface proteins antigen 43 and curli. Changes in gene expression caused by the deletion of cueO were essentially independent of SoxR and OxyR, the global regulators of oxidative stress response.

A new method for mercury removal.

A method is described for the removal of mercury from solution by using the off-gas produced from aerobic cultures of Klebsiella pneumoniae M426. Cells growing in Hg-supplemented medium produced a black precipitate containing mercury and sulphur. The ratio of Hg:S was determined as approximately 1:1 by analysis using proton-induced X-ray emission, suggesting precipitation of HgS within the culture. The outlet gases produced by a mercury-unsupplemented aerated culture were bubbled into an external chamber supplemented with up to 10 mg HgCl(2)/ml. A yellowish-white precipitate formed, corresponding to 99% removal of the mercury from solution within 120 min. Energy dispersive X-ray microanalysis showed that this metal precipitate consisted of mercury, carbon and sulphur. Formation of mercury carbonate was discounted since similar precipitation occurred at pH 2 and no oxygen was detected in the solid, which gave an X-ray powder pattern suggesting an amorphous material, with no evidence of HgS. Precipitation of mercury with a volatile organosulphur compound is suggested. Bio-precipitation of heavy metals by using culture off-gas is a useful approach because it can be used with concentrated or physiologically incompatible solutions. Since the metal precipitate is kept separate from the bacterial biomass, it can be managed independently.

A design for life: prokaryotic metal-binding MerR family regulators.

The MerR family of metal-binding, metal-responsive proteins is unique in that they activate transcription from unusual promoters and coordinate metals through cysteine (and in the case of ZntR, histidine) residues. They have conserved primary structures yet can effectively discriminate metals in vivo.

The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations.

The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal medium supplemented with elevated copper concentrations (as copper-glycine) has been analysed using whole-genome oligonucleotide microarrays. At 750 muM copper-glycine, the expression of both the cue and cus copper-export systems is evident. At near-lethal copper concentrations (2 mM copper-glycine), the expression of these two regulons increases significantly. Other regulons with increased transcription in response to elevated concentrations of copper-glycine include those for the superoxide stress response, iron homeostasis, and envelope stress. Furthermore, a variety of ORFs with decreased expression in response to increased copper-glycine has been identified, including the zinc ABC transporter and genes involved in the chemotactic response.

Climate factors influencing bacterial count in background air samples.

Total (as opposed to culturable) bacterial number counts are reported for four sites in the United Kingdom measured during campaigns over four separate seasons. These are interpreted in relation to simple climatic factors, i.e. temperature, wind speed and wind direction. Temperature has a marked effect at all four sites with data for a rural coastal site conforming best to a simple exponential model. Data for the other rural and urban locations show a baseline similar to that determined at the coastal rural location, but with some very significant positive excursions. The temperature dependence of bacterial number is found to conform to that typical of bacterial growth rates. At the coastal rural location, bacterial numbers normalised for temperature show no dependence on wind speed whilst at the inland sites there is a decrease with increasing wind speed of the form expected for a large area source. Only one site appeared to show a systematic relationship of bacterial concentrations to wind direction that being a site in the suburbs of Birmingham with highest number concentrations observed on a wind sector approaching from the city centre. PCR techniques have been used to identify predominant types of bacteria and results are presented which show that Bacillus was the dominant genus observed at the three inland sites during the winter and summer seasons. Pseudomonas appeared with comparable frequency at certain sites and seasons. There was in general a greater diversity of bacteria at the coastal site than at the inland sites.

Biosensors for detection of mercury in contaminated soils.

Biosensors based on whole bacterial cells and on bacterial heavy metal binding protein were used to determine the mercury concentration in soil. The soil samples were collected in a vegetable garden accidentally contaminated with elemental mercury 25 years earlier. Bioavailable mercury was measured using different sensors: a protein-based biosensor, a whole bacterial cell based biosensor, and a plant sensor, i.e. morphological and biochemical responses in primary leaves and roots of bean seedlings grown in the mercury-contaminated soil. For comparison the total mercury concentration of the soil samples was determined by AAS. Whole bacterial cell and protein-based biosensors gave accurate responses proportional to the total amount of mercury in the soil samples. On the contrary, plant sensors were found to be less useful indicators of soil mercury contamination, as determined by plant biomass, mercury content of primary leaves and enzyme activities.

The MerR family of transcriptional regulators.

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.

ZccR--a MerR-like regulator from Bordetella pertussis which responds to zinc, cadmium, and cobalt.

A transcriptional regulator of the MerR family encoded by Bordetella pertussis was characterized in Escherichia coli and in vitro. Uniquely, the regulator responded specifically to Zn(II), Cd(II), and Co(II) and was named ZccR. Gel shift assays confirmed that ZccR binds to an adjacent divergent promoter possessing an elongated spacer region of 19bp between the -10 and -35 elements, and that Zn(II), Co(II), and Cd(II) reduced the protein affinity for DNA. Site-directed mutagenesis of four cysteine and six histidine residues of ZccR showed that the cysteine residues at positions 77, 112, and 122, conserved in many of the metal-responsive MerR-like regulators, were essential for induction. Mutagenesis of the histidine residues (positions 73, 87, 90, 126, 140, and 142) revealed that histidine residues at 90, 140, and 142 were required for full induction by all three metals.

Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in enterobacteria from the preantibiotic era.

Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion elements.

The Escherichia coli copper-responsive copA promoter is activated by gold.

The copA gene of Escherichia coli encodes a copper transporter and its promoter is normally regulated by Cu(I) ions and CueR, a MerR-like transcriptional activator. We show that CueR can also be activated by gold salts and that Cys(112) and Cys(120) are involved in recognition of gold, silver, and copper salts. Gold activation is unaffected by copper chelating agents but is affected by general metal chelators. This is the first example of specific regulation of transcription by gold, and we briefly speculate that the biological effects of gold antiarthritic drugs may be through their effects on copper management in eukaryotic systems.

The Pco proteins are involved in periplasmic copper handling in Escherichia coli.

The interactions between the plasmid-borne copper resistance determinant, pco, and the main copper export system in Escherichia coli have been investigated and no direct interaction has been found. The PcoE and PcoC proteins are periplasmic and PcoC binds one Cu ion per protein molecule. PcoA is also periplasmic and can substitute for the chromosomally encoded CueO protein. The pco determinant is proposed to exert its effect through periplasmic handling of excess copper ions and to increase the level of resistance to copper ions above that conferred by copA alone.

A new chromosomal locus associated with gut-modulated phenotypes in Salmonella enterica serotype typhimurium.

A cosmid DNA library had been constructed previously from 40-kb fragments of genomic DNA from a virulent invasive strain of Salmonella enterica serotype Typhimurium (TML) in an avirulent hypo-invasive Typhimurium strain (LT7). Selection of invasive clones from the library was attempted by iterative passage through a rabbit ileal organ culture. After the fourth passage, a clone, designated LT7(pHC20-2), was isolated. Exposure to both gut tissue and Caco-2 cells enhanced the growth, invasiveness for gut and Caco-2 cells, and flagellin expression of LT7(pHC20-2) although its invasiveness was less than that of strain TML. Expression of appendages (surface structures c. 60-70 nm diameter) was shown to play a role in but not to confer invasiveness, and was demonstrated in the absence of direct contact with eukaryotic cells. Exposure to gut tissue also affected the expression of several outer-membrane proteins (OMPs) in all four Salmonella strains--TML, LT7, LT7(pHC79), LT7(pHC20 2)--used in this work. As the genes involved in flagella, invasin and porin expression are distributed around the salmonella chromosome, it is possible that pHC20-2 encodes a pleiotropic regulator of genes involved in gastro-enteritic virulence and adaptation to the in-vivo gut environment. pHC20-2 mapped at c. centisome 25 on the salmonella chromosome close to, but distinct from, SPI-5.

Invasiveness of Salmonella serotypes Typhimurium and Enteritidis of human gastro-enteritic origin for rabbit ileum: role of LPS, plasmids and host factors.

An organ culture system involving explants of distal rabbit ileum was used to study the roles of lipopolysaccharide (LPS) and plasmids in primary invasiveness for enterocytes in situ of strains of Salmonella serotypes Typhimurium and Enteritidis. Long-chain LPS per se does not confer invasiveness on Typhimurium, as known avirulent, hypo-invasive strains express smooth LPS. However, the invasiveness of a naturally occurring rough isogenic derivative of Salmonella serotype Enteritidis PT 4 was about half that of its wild-type parent. Therefore, smooth LPS appears to play a secondary role in maximising invasiveness. No evidence was found to correlate primary invasiveness for gut of 18 strains of Typhimurium with plasmid profiles in general or with the 60-MDa serovar-specific virulence plasmid in particular. Evidence is presented that strongly suggests a seasonal variability in susceptibility of rabbit gut to invasion by Typhimurium. Although no explanation is given for this summer insusceptibility, the data indicate the importance of the physiological status of the host in relation to susceptibility to invasion by Salmonella.

Selection and characterization of mercury-independent activation mutants of the Tn501 transcriptional regulator, MerR.

MerR is the transcriptional regulator of the mercury-resistance (mer) operon of transposon Tn501, acting at the mer promoter as both an activator in the presence of mercuric salts and a repressor in their absence. This paper reports a method for selection of constitutive activator mutants, which activate transcription in the absence of HgII, and the characterization of these MerRAC proteins. At least two mutations in the MerR protein were found necessary for strong mercury-independent activation, and these mutations lie in the C-terminal two-thirds of the MerR protein near the HgII-binding cysteines. A triple mutation was shown to increase activation over the corresponding double mutations. All mutant proteins caused further activation in the presence of HgII. The data support a mechanism in which a conformational change of one or both MerR subunits in the homodimer drives a distortion of DNA bound to a helix-turn-helix structure in the N-terminal region. A mutation in this putative helix-turn-helix region severely reduced both the repressor and activator functions of MerR.