scholarly journals Se(VI) Reduction and the Precipitation of Se(0) by the Facultative Bacterium Enterobacter cloacae SLD1a-1 Are Regulated by FNR

2007 ◽  
Vol 73 (6) ◽  
pp. 1914-1920 ◽  
Author(s):  
N. Yee ◽  
J. Ma ◽  
A. Dalia ◽  
T. Boonfueng ◽  
D. Y. Kobayashi
Keyword(s):  
Reductase Activity ◽  
Regulatory Gene ◽  
Enterobacter Cloacae ◽  
Elemental Selenium ◽  
Wild Type ◽  
E Coli ◽  
First Order ◽  
Mutant Strains ◽  
Reaction Constants ◽  
Genetic Regulator

ABSTRACT The fate of selenium in the environment is controlled, in part, by microbial selenium oxyanion reduction and Se(0) precipitation. In this study, we identified a genetic regulator that controls selenate reductase activity in the Se-reducing bacterium Enterobacter cloacae SLD1a-1. Heterologous expression of the global anaerobic regulatory gene fnr (fumarate nitrate reduction regulator) from E. cloacae in the non-Se-reducing strain Escherichia coli S17-1 activated the ability to reduce Se(VI) and precipitate insoluble Se(0) particles. Se(VI) reduction by E. coli S17-1 containing the fnr gene occurred at rates similar to those for E. cloacae, with first-order reaction constants of k = 2.07 × 10−2 h−1 and k = 3.36 × 10−2 h−1, respectively, and produced elemental selenium particles with identical morphologies and short-range atomic orders. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reductase activity and unable to precipitate elemental selenium. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest that Se(VI) reduction and the precipitation of Se(0) by facultative anaerobes are regulated by oxygen-sensing transcription factors and occur under suboxic conditions.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

1979 ◽  
Vol 179 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Jeffrey D. Hillman
Keyword(s):  
Escherichia Coli ◽  
Simple Procedure ◽  
Rabbit Antiserum ◽  
Double Diffusion ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glycolytic Intermediate ◽  
E Coli ◽  
Catalytic Function ◽  
Mutant Strains

NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

scholarly journals Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

2006 ◽  
Vol 72 (8) ◽  
pp. 5173-5180 ◽  
Author(s):  
Helen Ridley ◽  
Carys A. Watts ◽  
David J. Richardson ◽  
Clive S. Butler
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Selective Reduction ◽  
Enterobacter Cloacae ◽  
Anaerobic Growth ◽  
Elemental Selenium ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Distinct Membrane

ABSTRACT Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed V max of 500 nmol SeO4 2− min−1 mg−1 (k cat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

scholarly journals Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

2013 ◽  
Vol 79 (10) ◽  
pp. 3171-3175 ◽  
Author(s):  
Joanne Theisen ◽  
Gerben J. Zylstra ◽  
Nathan Yee
Keyword(s):  
Genetic Evidence ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Transport Pathway ◽  
Content Type ◽  
E Coli ◽  
Reduction Activity ◽  
Mutant Strains ◽  
Molybdate Transport ◽  
K 12

ABSTRACTThe genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction inEscherichia coliK-12. The results show that mutants deleted of themoaA,moaB,moaE, ormoggene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of themodBormodCgene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction inE. coliinvolves a molybdoenzyme.

EnteropathogenicEscherichia coliinhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

2006 ◽  
Vol 290 (1) ◽  
pp. G30-G35 ◽  
Author(s):  
Alip Borthakur ◽  
Ravinder K. Gill ◽  
Kim Hodges ◽  
Krishnamurthy Ramaswamy ◽  
Gail Hecht ◽  
...  
Keyword(s):  
Surface Expression ◽  
Monocarboxylate Transporter ◽  
Wild Type ◽  
Infantile Diarrhea ◽  
Monocarboxylate Transporter 1 ◽  
E Coli ◽  
Food Borne ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Fatty Acid Absorption

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+and Cl−/HCO3−exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. III EVOLUTION OF THE ebg REPRESSOR DURING SELECTION FOR ENHANCED LACTASE ACTIVITY

Genetics ◽  
1977 ◽  
Vol 85 (2) ◽  
pp. 193-201
Author(s):  
Barry G Hall ◽  
Norma D Clarke
Keyword(s):  
Regulatory Gene ◽  
Enzyme Synthesis ◽  
Lactase Activity ◽  
Wild Type ◽  
Regulatory Mutation ◽  
E Coli ◽  
Lactose Induction ◽  
A Cell ◽  
Lactose Utilization ◽  
Selection For

ABSTRACT The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg  + strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

scholarly journals Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

2008 ◽  
Vol 191 (5) ◽  
pp. 1472-1479 ◽  
Author(s):  
Jinki Yeom ◽  
Che Ok Jeon ◽  
Eugene L. Madsen ◽  
Woojun Park
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Ferric Reductase ◽  
Wild Type ◽  
Cell Extracts ◽  
Mutant Strains ◽  
First Time

ABSTRACT Pseudomonas putida harbors two ferredoxin-NADP+ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k cat/Km value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

2015 ◽  
Vol 25 (6) ◽  
pp. 394-402 ◽  
Author(s):  
Taylor L. Fischer ◽  
Robert J. White ◽  
Katherine F.K. Mares ◽  
Devin E. Molnau ◽  
Justin J. Donato
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
E Coli ◽  
Enoyl Acp Reductase ◽  
Selection For

<b><i>Background/Aims:</i></b> We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in <i>Escherichia coli</i>. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. <b><i>Methods:</i></b> ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into <i>E. coli</i>, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. <b><i>Results:</i></b> Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into <i>E. coli</i>, whereas the mutant remained susceptible to triclosan<i>. </i>Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. <b><i>Conclusion:</i></b> ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

scholarly journals Vitamin B12 in photosynthetic bacteria and methionine synthesis by Rhodopseudomonas spheroides

1967 ◽  
Vol 102 (3) ◽  
pp. 774-781 ◽  
Author(s):  
S. E. Cauthen ◽  
J. R. Pattison ◽  
J Lascelles
Keyword(s):  
Photosynthetic Bacteria ◽  
Methylenetetrahydrofolate Reductase ◽  
Reductase Activity ◽  
Vitamin B ◽  
Wild Type ◽  
E Coli ◽  
Vitamin B 12 ◽  
Rhodopseudomonas Spheroides ◽  
Methionine Synthesis ◽  
Dependent Pathway

1. Assay of some photosynthetic bacteria for vitamin B(12) showed them to be relatively rich in this factor. Rhodopseudomonas spheroides, grown photosynthetically in Co(2+)-supplemented medium, contained about 100mug./g. dry wt. 2. Extracts of wild-type Rps. spheroides methylated homocysteine by a mechanism similar to the cobalamin-dependent pathway present in Escherichia coli. However, no mechanism similar to the cobalamin-independent N(5)-methyltetrahydrofolate-homocysteine transmethylase of E. coli could be detected in Rps. spheroides. 3. N(5)N(10)-Methylenetetrahydrofolate-reductase activity was found in Rps. spheroides. 4. A methionine-requiring mutant strain of Rps. spheroides (strain 2/33), which does not respond to homocysteine, made the same amount of vitamin B(12) as the parent organism. Extracts did not form methionine from N(5)-methyltetrahydrofolate and homocysteine even in the presence of cofactors shown to be necessary with the parent strain, and it is concluded that the mutant is blocked in the formation of the apoenzyme of a homocysteine-methylating system similar to the vitamin B(12)-dependent one in E. coli.

scholarly journals The Escherichia coli O157 Flagellar Regulatory Gene flhC and Not the Flagellin Gene fliC Impacts Colonization of Cattle

2006 ◽  
Vol 74 (5) ◽  
pp. 2894-2905 ◽  
Author(s):  
Heather S. Dobbin ◽  
Carolyn J. Hovde ◽  
Christopher J. Williams ◽  
Scott A. Minnich
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Oral Dose ◽  
Regulatory Gene ◽  
Wild Type ◽  
E Coli ◽  
Regulatory Mutant ◽  
The Relationship ◽  
Better Than

ABSTRACT A virulent European Escherichia coli O157:H − isolate is nonmotile due to a 12-bp deletion in the flagellar regulatory gene flhC. To investigate the contribution of flhC in the relationship between E. coli O157:H7 and cattle, we constructed a similar flhC regulatory mutant in the well-characterized strain ATCC 43894. There was no difference in the growth rate between the wild type and this regulatory mutant, but phenotypic arrays showed substrate utilization differences. Survival in the bovine gastrointestinal tract and colonization of the rectoanal junction mucosa were assessed. Mixtures of both strains were given orally or rectally to steers or administered into the rumen of cattle dually cannulated at the rumen and duodenum. One day post-oral dose, most rectal/fecal isolates (74%) were the regulatory mutant, but by 3 days post-oral dose and throughout the 42-day experiment, ≥80% of the isolates were wild type. Among steers given a rectal application of both strains, wild-type isolates were the majority of isolates recovered on all days. The regulatory mutant survived better than the wild type in both the rumen and duodenum. To test the role of motility, a filament mutant (ΔfliC) was constructed and similar cattle experiments were performed. On all days post-oral dose, the majority of isolates (64% to 98%) were the filament mutant. In contrast, both strains were recovered equally post-rectal application. Thus, the regulatory mutant survived passage through the bovine gastrointestinal tract better than the wild type but failed to efficiently colonize cattle, and the requirement of flhC for colonization was not dependent on a functional flagellum.

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