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.

scholarly journals The Secreted Pyomelanin Pigment of Legionella pneumophila Confers Ferric Reductase Activity

2007 ◽  
Vol 75 (8) ◽  
pp. 4062-4070 ◽  
Author(s):  
Christa H. Chatfield ◽  
Nicholas P. Cianciotto
Keyword(s):  
Growth Medium ◽  
Legionella Pneumophila ◽  
Reductase Activity ◽  
Inhibitory Effect ◽  
Homogentisic Acid ◽  
Ferric Reductase ◽  
Wild Type ◽  
Reduction Of Iron ◽  
Strong Inhibitory Effect ◽  
First Time

ABSTRACT The virulence of Legionella pneumophila is dependent upon its capacity to acquire iron. To identify genes involved in expression of its siderophore, we screened a mutagenized population of L. pneumophila for strains that were no longer able to rescue the growth of a ferrous transport mutant. However, an unusual mutant was obtained that displayed a strong inhibitory effect on the feoB mutant. Due to an insertion in hmgA that encodes homogentisate 1,2-dioxygenase, the mutant secreted increased levels of pyomelanin, the L. pneumophila pigment that is derived from secreted homogentisic acid (HGA). Thus, we hypothesized that L. pneumophila-secreted HGA-melanin has intrinsic ferric reductase activity, converting Fe3+ to Fe2+, but that hyperpigmentation results in excessive reduction of iron that can, in the case of the feoB mutant, be inhibitory to growth. In support of this hypothesis, we demonstrated, for the first time, that wild-type L. pneumophila secretes ferric reductase activity. Moreover, whereas the hyperpigmented mutant had increased secreted activity, an lly mutant specifically impaired for pigment production lacked the activity. Compatible with the nature of HGA-melanins, the secreted ferric reductase activity was positively influenced by the amount of tyrosine in the growth medium, resistant to protease, acid precipitable, and heterogeneous in size. Together, these data represent the first demonstration of pyomelanin-mediated ferric reduction by a pathogenic bacterium.

scholarly journals Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

1999 ◽  
Vol 65 (6) ◽  
pp. 2631-2635 ◽  
Author(s):  
Sonja Isken ◽  
Antoine Derks ◽  
Petra F. G. Wolffs ◽  
Jan A. M. de Bont
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Organic Solvents ◽  
Critical Concentration ◽  
Bacterial Membrane ◽  
Maximal Growth ◽  
Wild Type ◽  
Active Efflux ◽  
Maximal Growth Rate ◽  
Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

scholarly journals Identification of the NADH-oxidase gene in Trichomonas vaginalis

2019 ◽  
Vol 119 (2) ◽  
pp. 683-686 ◽  
Author(s):  
Aline Lamien-Meda ◽  
David Leitsch
Keyword(s):  
Oxidase Activity ◽  
Trichomonas Vaginalis ◽  
Nadh Oxidase ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Oxidase Gene ◽  
Reading Frame ◽  
Sexually Transmitted ◽  
Cell Extracts ◽  
Nadh Oxidase Activity

AbstractThe microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.

Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid

2001 ◽  
Vol 47 (3) ◽  
pp. 222-228 ◽  
Author(s):  
Anne J Anderson ◽  
Charles D Miller
Keyword(s):  
Hydrogen Peroxide ◽  
Pseudomonas Putida ◽  
Peracetic Acid ◽  
Catalase Activity ◽  
Specific Activity ◽  
Active Oxygen Species ◽  
Oxygen Species ◽  
Deficient Mutant ◽  
Wild Type ◽  
Cell Extracts

Peracetic acid is used as a sterilant in several industrial settings. Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A. Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts. Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid. The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture. Amendment of sterilized soils with wild-type P. putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant. The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid. No differential recovery of the wild-type and catalase A mutant of P. putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.Key words: Pseudomonas putida (Pp), activated oxygen species (AOS), hydrogen peroxide, luciferase, colonization.

scholarly journals Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources

2005 ◽  
Vol 71 (10) ◽  
pp. 6104-6114 ◽  
Author(s):  
D. J. Koch ◽  
C. Rückert ◽  
D. A. Rey ◽  
A. Mix ◽  
A. Pühler ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Gene Products ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
Similarity Searches

ABSTRACT Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

scholarly journals Mutant strains of Escherichia coli lacking global regulators, arcA and fis, demonstrate better growth fitness by pathway reprogramming under acetate metabolism

2021 ◽  
Author(s):  
Shikha Jindal ◽  
Mahesh S. Iyer ◽  
Poonam Jyoti ◽  
Shyam Kumar Masakapalli ◽  
K V Venkatesh
Keyword(s):  
Growth Rate ◽  
Metabolic Flux ◽  
Microbial Metabolism ◽  
Metabolic Reprogramming ◽  
Acetate Metabolism ◽  
Flux Analysis ◽  
Wild Type ◽  
Global Regulators ◽  
Acetate Uptake ◽  
Mutant Strains

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A systems-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the substrates available in a range of nutrient niches such as the mammalian gut and high-fat diet. Therefore, investigating the study on acetate metabolism is highly significant. It is well known that the global regulators arcA and fis regulate acetate uptake genes in E. coli under glucose condition. In this study, we deciphered the growth and flux distribution of E.coli transcription regulatory knockout mutants ΔarcA, Δfis and double deletion mutant, ΔarcAfis under acetate using 13C-Metabolic Flux Analysis which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~1.2-1.6 fold) with a proportionate increase in acetate uptake rates compared to the wild-type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes, which conferred an advantage of faster growth with better carbon usage in all the mutants. Under acetate metabolism, the mutants exhibited higher fluxes in the TCA cycle (~18-90%) and lower gluconeogenesis flux (~15-35%) with the proportional increase in growth rate. This study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate to acetyl-CoA and therefore are potential candidates that can serve as a precursor for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products.

scholarly journals Influence of Ethylene Signaling in the Crosstalk Between Fe, S, and P Deficiency Responses in Arabidopsis thaliana

2021 ◽  
Vol 12 ◽  
Author(s):  
María José García ◽  
Macarena Angulo ◽  
Carlos García ◽  
Carlos Lucena ◽  
Esteban Alcántara ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Reductase Activity ◽  
Nutrient Deficiency ◽  
Fe Deficiency ◽  
Energy Costs ◽  
Ethylene Signaling ◽  
Ferric Reductase ◽  
Wild Type ◽  
P Deficiency ◽  
Specific Manner

To cope with P, S, or Fe deficiency, dicot plants, like Arabidopsis, develop several responses (mainly in their roots) aimed to facilitate the mobilization and uptake of the deficient nutrient. Within these responses are the modification of root morphology, an increased number of transporters, augmented synthesis-release of nutrient solubilizing compounds and the enhancement of some enzymatic activities, like ferric reductase activity (FRA) or phosphatase activity (PA). Once a nutrient has been acquired in enough quantity, these responses should be switched off to minimize energy costs and toxicity. This implies that they are tightly regulated. Although the responses to each deficiency are induced in a rather specific manner, crosstalk between them is frequent and in such a way that P, S, or Fe deficiency can induce responses related to the other two nutrients. The regulation of the responses is not totally known but some hormones and signaling substances have been involved, either as activators [ethylene (ET), auxin, nitric oxide (NO)], or repressors [cytokinins (CKs)]. The plant hormone ET is involved in the regulation of responses to P, S, or Fe deficiency, and this could partly explain the crosstalk between them. In spite of these crosslinks, it can be hypothesized that, to confer the maximum specificity to the responses of each deficiency, ET should act in conjunction with other signals and/or through different transduction pathways. To study this latter possibility, several responses to P, S, or Fe deficiency have been studied in the Arabidopis wild-type cultivar (WT) Columbia and in some of its ethylene signaling mutants (ctr1, ein2-1, ein3eil1) subjected to the three deficiencies. Results show that key elements of the ET transduction pathway, like CTR1, EIN2, and EIN3/EIL1, can play a role in the crosstalk among nutrient deficiency responses.

scholarly journals Purification and characterization of morphinone reductase from Pseudomonas putida M10

1994 ◽  
Vol 301 (1) ◽  
pp. 97-103 ◽  
Author(s):  
C E French ◽  
N C Bruce
Keyword(s):  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Apparent Homogeneity ◽  
Ping Pong

The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.

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.

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