Anaerobic regulation by an atypical Arc system in Shewanella oneidensis

Shewanella oneidensis strain MR-1 is well known for its respiratory versatility, yet little is understood about how it regulates genes involved in anaerobic respiration. The Arc two-component system plays an important role in this process in Escherichia coli; therefore, we determined its function in S. oneidensis. arcA from S. oneidensis complements an E. coli arcA mutant, but the Arc regulon in S. oneidensis constitutes a different suite of genes. For example, one of the strongest ArcA-regulated gene clusters in E. coli, sdh, is not regulated by the Arc system in S. oneidensis, and the cyd locus, which is induced by ArcA in E. coli under microaerobic conditions, is repressed by ArcA in S. oneidensis under anaerobic conditions. One locus that we identified as being potentially regulated by ArcA in S. oneidensis contains genes predicted to encode subunits of a dimethyl sulphoxide (DMSO) reductase. We demonstrate that these genes encode a functional DMSO reductase, and that an arcA mutant cannot fully induce their expression and is defective in growing on DMSO under anaerobic conditions. While S. oneidensis lacks a highly conserved full-length ArcB homologue, ArcA is partially activated by a small protein homologous to the histidine phosphotransfer domain of ArcB from E. coli, HptA. This protein alone is unable to compensate for the lack of arcB in E. coli, indicating that another protein is required in addition to HptA to activate ArcA in S. oneidensis.     

Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli

Microbial cells possess numerous sensing/regulator systems in order to respond rapidly to environmental changes. Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. A group of global regulators, which include the one component Fnr protein and the two-component Arc system, coordinate the adaptive responses. To quantitate the contribution of Arc and FNR-dependent regulation under microaerobic conditions, the gene expression pattern of the electron transfer chain genes and the TCA cycle genes in wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that the TCA cycle genes, icd, gltA, sucC, and sdhC are repressed by ArcA while Fnr has a minor or no effect on the expression of these genes under microaerobic conditions. The expression levels of the electron transfer chain genes, nuoA, ndh, and ubiE, were not significantly affected by either ArcA or Fnr regulation proteins, while a lower expression of cydA (up to 9-fold lower) and a higher expression of cyoA (up to 31-fold higher) were observed in cultures of the arcA mutant strain compared to those of the wild type. Since significantly higher NADH/NAD+ ratios were previously observed in cultures of the arcA mutant strain compared to the wild type it seems that the cytochrome o oxidase (the product of cyoABCDE) cannot efficiently support aerobic respiration when the cells are grown under microaerobic conditions.     

Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. The adaptive responses are coordinated by a group of global regulators, which include the one-component Fnr protein, and the two-component Arc system. To quantitate the contribution of Arc and FNR dependent regulation under microaerobic conditions, the gene expression pattern of the fnr the arcA and arcB regulator genes, and the glycolysis related genes in a wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that ArcA has a negative effect on fnr expression under microaerobic conditions. Moreover, the expression levels of the FNR regulated genes, yfiD and frdA, were higher in cultures of the arcA mutant strain compared to the wild-type. These imply that a higher level of the FNR regulator is in the activated form in cultures of the arcA mutant strain compared to the wild-type during the transition from aerobic to microanaerobic growth. The results also show that the highest expression level of aceE, pflB, and adhE were obtained in cultures of the arcA mutant strain under microaerobic growth while higher levels of ldhA expression were obtained in cultures of the arcA mutant strain and the arcA, fnr double mutant strain compared to the wild-type and the fnr mutant strain. While the highest expression of adhE and pflB in cultures of the arcA mutant strain can explain the previous report of high ethanol flux and flux through pyruvate formate lyase (PFL) in cultures of this strain, the higher level of ldhA expression was not sufficient to explain the trend in lactate fluxes. The results indicate that lower conversion of pyruvate to acetyl-CoA is the main reason for high fluxes through lactate dehydrogenase (LDH) in cultures of the arcA, fnr double mutant strain.     

A second global regulator gene (arcB) mediating repression of enzymes in aerobic pathways of Escherichia coli.

In Escherichia coli anaerobic growth lowers the basal or induced levels of numerous enzymes associated with aerobic metabolism. Mutations in arcA (dye) at min 0 relieve this pleiotropic anaerobic repression and render the cell sensitive to the redox dye toluidine blue. In this study we identified a second pleiotropic control gene, arcB, at min 69.5. Mutations, including a deletion, in this gene also relieved the anaerobic repression and caused sensitivity to toluidine blue. Mutations in arcA or arcB did not significantly change the catabolite repression of the target phi(sdh-lacZ) operon, in which lacZ is fused to a structural gene for succinate dehydrogenase, nor did the mutations strikingly influence the pattern of excretion products during glucose fermentation. The presence of arcA+ in a multicopy plasmid restored anaerobic repression in arcB mutants, as indicated by the expression of phi(sdh-lacZ). The arcB product might be a sensor protein for the redox or energy state of the arc regulatory system.     

Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation.

In Escherichia coli, mutations in arcA (dye) or arcB anaerobically derepress the synthesis of a multitude of enzymes of aerobic function, and mutations in arcA or cpxA impair F-pilus formation. It is thought that arcA encodes a promoter-recognizing protein, whereas arcB and cpxA encode sensor proteins which interact with the arcA product. In this study we found that anaerobic growth of a wild-type F' strain decreased the synthesis of both the enzymes and the pilus. Although the two arcA mutants examined were both anaerobically derepressed in the enzymes and impaired in aerobic pilus formation as expected, one mutant hyperproduced the pilus anaerobically. The two arcB mutants examined showed normal pilus formation when grown aerobically. When grown anaerobically they developed more pili than the wild-type strain did when grown aerobically. When a cpxA mutant was examined for synthesis of two aerobic enzymes, normal regulation was found. The available data suggest the following. The arcA product anaerobically represses certain genes of aerobic function and activates certain genes related to F function. It appears that the arcB product senses the redox or energy state; absence of the gene function shifts the arcA product to the nonrepressive form for enzyme synthesis for aerobic pathways. The cpxA product, on the other hand, senses the sexual state; absence of the gene function shifts the arcA product to the inactive form for F-pilus synthesis.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.     

Specific induction and carbon/nitrogen repression of arginine catabolism gene of Aspergillus nidulans--functional in vivo analysis of the otaA promoter

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp).We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.     

Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli

The cgtA gene codes for a common GTP-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. Although cgtA is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. In Escherichia coli, dysfunction or overexpression of the cgtA gene causes problems in various chromosomal functions, like synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round. It is not know how the cgtA gene product regulates these processes. Here we investigated effects of cgtA dysfunction on replication of plasmid and phage replicons. We found that replication of some plasmids (e.g., ColE1-like) is not affected in the cgtA mutant. On the other hand, dysfunction of the cgtA gene caused a strong inhibition of lambda plasmid DNA replication. Bacteriophage lambda development was severely impaired in the cgtA mutant. Replication of other plasmid replicons (derivatives of F, R1, R6K, and RK2) was influenced by the cgtA mutation moderately. It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression.     

Genetic analysis of Escherichia coli K-12 chromosomal mutants defective in expression of F-plasmid functions: identification of genes cpxA and cpxB.

Two temperature-sensitive, chromosomal mutants of Escherichia coli were selected for their inability to express deoxyribonucleic acid donor activity and other activities associated with the conjugative plasmid F. These mutants were also auxotrophic for isoleucine and valine at 41 degrees C. Each mutant strain contained two altered genes: cpxA, located at 88 min on the E. coli K-12 genetic map, and cpxB, located at 41 min. Mutations in both genes were required for maximal expression of mutant phenotypes. The parent strain of mutants KN401 and KN312 already contained the cpxB mutation that is present in both mutants (cpxB1). This mutation by itself was cryptic. The cpxA mutations represent different mutant alleles since they are of independent origin. A cpxA mutation by itself significantly affected the expression of plasmid functions and growth at 41 degrees C in the absence of isoleucine and valine, but strains containing both a cpxA and cpxB mutation were more severely affected. Along with the observation that both cpxA mutations were revertable, the temperature sensitivity of cpxA cpxB+ cells suggests that both cpxA alleles contain point mutations that do not completely destroy the activity of the cpxA gene product.     

山梨糖脱氢酶和氨甲酰化酶的融合表达

利用大肠杆菌-生黑醋杆菌穿梭载体pUG18构建了山梨糖脱氢酶基因sdh和氨甲酰化酶基因hyuC的融合表达质粒pUG18-SDH-HyuC.在JM109/pUG18-SDH-HyuC中检测到山梨糖脱氢酶(SDH)和氨甲酰化酶(HyuC)的酶活性,大量的SDH和HyuC不以融合蛋白形式存在.将质粒pUG18-SDH-HyuC电转化至生黑醋杆菌H24,检测到HyuC活性,但尚未检测到SDH酶活性.从生黑醋杆菌转化子中提取质粒,重新转化至JM109,又可同时检测到SDH和HyuC活性,传代实验和对质粒的鉴定结果也说明该质粒在H24中稳定存在.     

The muc genes of pKM101 are induced by DNA damage

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.     

Growth recovery on glucose under aerobic conditions of an Escherichia coli strain carrying a phosphoenolpyruvate:carbohydrate phosphotransferase system deletion by inactivating arcA and overexpressing the genes coding for glucokinase and galactose permease

In Escherichia coli the phosphotransferase system (PTS) consumes one molecule of phosphoenolpyruvate (PEP) to phosphorylate each molecule of internalized glucose. PEP bioavailability into the aromatic pathway can be increased by inactivating the PTS. However, the lack of the PTS results in decreased glucose transport and growth rates. To overcome such drawbacks in a PTS(-) strain and reconstitute rapid growth on glucose phenotype (Glc(+)), the glk and galP genes were cloned into a plasmid and the arcA gene was inactivated. Simultaneous overexpression of glk and galP increased the growth rate and regenerated a Glc(+) phenotype. However, the highest growth rate was obtained when glk and galP were overexpressed in the arcA(-) background. These results indicated that the arcA mutation enhanced glycolytic and respiratory capacities of the engineered strain.     

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed ( finO  ⁻) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10⁷-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ , traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.