Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12

Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.     

Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop

The CusSR two-component system (TCS) is a copper-sensing apparatus of E. coli that is responsible for regulating the copper-related homeostatic system. The dynamic characteristics of the CusSR network were modified by the introduction of a positive feedback loop. To construct the feedback loop, the CusR, which is activated by the cusC promoter, was cloned downstream of the cusC promoter and reporter protein. The feedback loop system, once activated by environmental copper, triggers the activation of the cusC promoter, which results in the amplification of a reporter protein and CusR expression. The threshold copper concentration for the activation of the modified CusSR TCS network was lowered from 2,476.5?μg/l to 247.7?μg/l, which indicates a tenfold increase in sensitivity. The intensity of the output signal was increased twofold, and was maintained for 16?h. The strategy proposed in this study can also be applied to modify the dynamic characteristics of other TCSs.     

Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria

Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli , the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP . A Δ cueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Δ cus E. coli . We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.     

Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR

Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbI as cueR for the Cu efflux regulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response.     

西瓜食酸菌抗铜基因cueR的生物信息学分析及功能验证

【背景】CueR被证实在模式细菌大肠杆菌的Cue抗铜系统中参与转录调控,西瓜食酸菌(Acidovorax citrulli)中是否有类似的机制尚不清楚。【目的】鉴定西瓜食酸菌中的cueR基因、分析其编码蛋白的特点与功能,可以为进一步探究类Cue系统在西瓜食酸菌铜稳态中的作用机制奠定基础。【方法】以大肠杆菌等4个模式细菌中已经鉴定的CueR为参照,运用生物信息学手段对西瓜食酸菌的CueR (AcCueR)与大肠杆菌的EcCueR、铜绿假单胞菌的PaCueR、沙门氏菌的SeCueR、霍乱弧菌的VcCueR蛋白进行结构、性质、亚细胞定位、互作因子等特征分析;利用同源重组插入突变技术构建西瓜食酸菌FC440菌株cueR基因的突变体,并制备突变体基因功能互补菌株,比较分析各菌株抗铜性表型。【结果】西瓜食酸菌和铜绿假单胞菌的CueR序列相似性最高;5个细菌的CueR蛋白均属于HTH-MerR-SF超家族,三级结构主要由α-螺旋和无规则卷曲构成;5种蛋白结构相似;AcCueR可以与西瓜食酸菌中P型ATP酶(即CopA)、多铜氧化酶CueO产生互作,且copA启动子中存在一个与CueR结合的回文结构。在含Cu~(2+)培养基上,突变菌株FC440(?cueR)生长能力明显减弱,基因功能互补菌株FC440(?cueR-cueR)的生长能力则完全恢复。【结论】西瓜食酸菌中的cueR基因与菌的抗铜性相关,其AcCueR蛋白与大肠杆菌等菌中的CueR具有相似的结构与功能,在西瓜食酸菌中可能存在类似于大肠杆菌的Cue抗铜系统。     

Measurement of cytoplasmic copper,silver, and gold with a lux biosensor shows copper and silver,but not gold,efflux by the CopA ATPase of Escherichia coli

Copper, silver, gold and other heavy metals are potentially toxic to cells. Copper is also essential and cellular levels must be carefully controlled. In contrast, there is no known biological role for silver or gold and they have not been recognized as metals that are under homeostatic control. Using a luminescent biosensor based of the Vibrio fischeri lux gene cluster under the control of the Escherichia coli copA promoter/CueR metal-responsive regulator, we could show that in E. coli, cytoplasmic copper and silver, but not gold, are regulated by the CopA ATPase, the major copper efflux pump.     

Copper sensitivity of cueO mutants of Escherichia coli K-12 and the biochemical suppression of this phenotype

The cueO gene of Escherichia coli encodes a multi-copper oxidase, which contributes to copper tolerance in this bacterium. It was observed that a cueO mutant was highly sensitive to killing by copper ions when cells were grown on defined minimal media. Copper sensitivity was correlated with accumulation of copper in the mutant strain. Growth of the cueO mutant in the presence of copper could be restored by addition of divalent zinc and manganese ions or ferrous iron but not by other first row transition metal ions or magnesium ions. Copper toxicity towards a cueO mutant could also be suppressed by addition of the superoxide quencher 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron), suggesting that a primary cause of copper toxicity is the copper-catalyzed production of superoxide anions in the cytoplasm.     

Characterization of copper-inducible promoters regulated by CpxA/CpxR in Escherichia coli

The copper stimulon in Escherichia coli consists of four regulons, the CueR-, CusS/CusR-, CpxA/CpxR-, and YedV/YedW regulons. E. coli mutants defective in cpxRA showed higher sensitivity to copper than the wild type. A total of 15 promoters were found to be induced in E. coli culture upon exposure to copper in a CpxA/CpxR-dependent manner. After gel-shift and DNase I foot-printing analyses, a conserved tandem repeat of pentanucleotide sequence, GTAAA(N)(4-8)GTAAA, with a conserved A of 4-bp upstream of each pentamer, was identified to be the CpxR-binding site. The difference in the orientation and location of the CpxR box is discussed with respect to the regulation mechanism among CpxR-regulon genes.     

The Pho regulons of bacteria

Bacterial Pho regulons contain genes whose expression is regulated by a specific two-component signal transduction system. The products of these genes are involved in the transport and assimilation of inorganic phosphate. The paper summarizes data on the organization and function of Pho regulons in gram-negative and gram-positive bacteria, with particular emphasis on the Pho regulons of the best studied bacteria Escherichia coli and Bacillus subtilis.     

Indole induces the expression of multidrug exporter genes in Escherichia coli

Our comprehensive expression cloning studies previously revealed that 20 intrinsic xenobiotic exporter systems are encoded in the Escherichia coli chromosome, but most of them are not expressed under normal conditions. In this study, we investigated the compounds that induce the expression of these xenobiotic exporter genes, and found that indole induces a variety of xenobiotic exporter genes including acrD, acrE, cusB, emrK, mdtA, mdtE and yceL. Indole treatment of E. coli cells confers rhodamine 6G and SDS resistance through the induction of mdtEF and acrD gene expression respectively. The induction of mdtE by indole is independent of the EvgSA two-component signal transduction system that regulates the mdtE gene, but mediated by GadX. On the other hand, the induction of acrD and mdtA was mediated by BaeSR and CpxAR, two-component systems. Interestingly, CpxAR system-mediated induction required intrinsic baeSR genes, whereas BaeSR-mediated induction was observed in the cpxAR gene-deletion mutant. BaeR and CpxR directly bound to different sequences of the acrD and mdtA promoter regions. These observations indicate that BaeR is a primary regulator, and CpxR enhances the effect of BaeR.