scholarly journals Formate-Dependent H2 Production by the Mesophilic Methanogen Methanococcus maripaludis

2008 ◽  
Vol 74 (21) ◽  
pp. 6584-6590 ◽  
Author(s):  
Boguslaw Lupa ◽  
Erik L. Hendrickson ◽  
John A. Leigh ◽  
William B. Whitman
Keyword(s):  
Dry Weight ◽  
Maximal Activity ◽  
Growth Conditions ◽  
H2 Production ◽  
Resting Cells ◽  
Wild Type ◽  
Methanococcus Maripaludis ◽  
Gene Encoding ◽  
Major Pathway ◽  
Key Enzyme

ABSTRACT Methanococcus maripaludis, an H2- and formate-utilizing methanogen, produced H2 at high rates from formate. The rates and kinetics of H2 production depended upon the growth conditions, and H2 availability during growth was a major factor. Specific activities of resting cells grown with formate or H2 were 0.4 to 1.4 U�mg−1 (dry weight). H2 production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (Kf ) was 3.6 mM. In contrast, in H2-grown cells this process followed sigmoidal kinetics, and the Kf was 9 mM. A key enzyme for formate-dependent H2 production was formate dehydrogenase, Fdh. H2 production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H2 production by a mutant containing a deletion of the coenzyme F420-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H2 production comprised Fdh1 and Fru. Because a Δfru-Δfrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F420-dependent methylene-H4MTP dehydrogenase (Mtd) or the H2-forming methylene-H4MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H2 production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H2 production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H2 production demonstrated the potential of M. maripaludis for the microbial production of H2 from formate.

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

scholarly journals Role of Acinetobacter baylyi Crc in Catabolite Repression of Enzymes for Aromatic Compound Catabolism

2009 ◽  
Vol 191 (8) ◽  
pp. 2834-2842 ◽  
Author(s):  
Tina Zimmermann ◽  
Tobias Sorg ◽  
Simone Yasmin Siehler ◽  
Ulrike Gerischer
Keyword(s):  
Catabolite Repression ◽  
Carbon Sources ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Wild Type ◽  
Acinetobacter Baylyi ◽  
Transcript Stability ◽  
Key Enzyme ◽  
In The Wild ◽  
First Time

ABSTRACT Here, we describe for the first time the Crc (catabolite repression control) protein from the soil bacterium Acinetobacter baylyi. Expression of A. baylyi crc varied according to the growth conditions. A strain with a disrupted crc gene showed the same growth as the wild type on a number of carbon sources. Carbon catabolite repression by acetate and succinate of protocatechuate 3,4-dioxygenase, the key enzyme of protocatechuate breakdown, was strongly reduced in the crc strain, whereas in the wild-type strain it underwent strong catabolite repression. This strong effect was not based on transcriptional regulation because the transcription pattern of the pca-qui operon (encoding protocatechuate 3,4-dioxygenase) did not reflect the derepression in the absence of Crc. pca-qui transcript abundance was slightly increased in the crc strain. Lack of Crc dramatically increased the mRNA stability of the pca-qui transcript (up to 14-fold), whereas two other transcripts (pobA and catA) remained unaffected. p-Hydroxybenzoate hydroxylase activity, encoded by pobA, was not significantly different in the absence of Crc, as protocatechuate 3,4-dioxygenase was. It is proposed that A. baylyi Crc is involved in the determination of the transcript stability of the pca-qui operon and thereby effects catabolite repression.

scholarly journals A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionineβ-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
João B. Vicente ◽  
Henrique G. Colaço ◽  
Francesca Malagrinò ◽  
Paulo E. Santo ◽  
André Gutierres ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Redox Homeostasis ◽  
Crystallographic Structure ◽  
Wild Type ◽  
Gene Encoding ◽  
Cellular Redox ◽  
Transsulfuration Pathway ◽  
Key Enzyme ◽  
A Minor ◽  
Ferrous Heme

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionineβ-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated byS-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenicCBSmutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.

scholarly journals Overexpression of Mannitol-1-Phosphate Dehydrogenase Increases Mannitol Accumulation and Adds Protection Against Chilling Injury in Petunia

2005 ◽  
Vol 130 (4) ◽  
pp. 605-610 ◽  
Author(s):  
Yu-Jen Chiang ◽  
C. Stushnoff ◽  
A.E. McSay ◽  
M.L. Jones ◽  
H.J. Bohnert
Keyword(s):  
Chilling Stress ◽  
Chilling Injury ◽  
Dry Weight ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Horticultural Crops ◽  
Transgenic Lines ◽  
And Control

Petunia ×hybrida (Hook) Vilm. cv. Mitchell was transformed with an E. coli gene encoding mannitol-1-phosphate dehydrogenase (mtlD). Four plant lines that grew on kanamycin and contained the mtlD transgene were identified. Two of these lines contained high levels of mannitol [high-mannitol lines M3 and M8; mean mannitol = 3.39 μmol·g-1 dry weight (DW)] compared to nontransformed wild-type plants (0.86 μmol·g-1 DW), while two lines had mannitol levels similar to wild-type plants (low-mannitol lines M2 and M9; mean mannitol = 1.05 μmol·g-1 DW). Transgenic and control plants were subjected to chilling stress (3 ± 0.5 °C day/0 ± 0.5 °C night, 12-hour photoperiod and 75% relative humidity) to evaluate the role of mannitol in chilling tolerance. Based upon foliage symptoms and membrane leakage after a 3-week chilling treatment, the high-mannitol containing lines, M3 and M8, were more tolerant of chilling stress than the low-mannitol containing transgenic lines, M2 and M9, and wild-type. Under nonchilling conditions mannitol was the only carbohydrate that differed among transgenic lines, but all carbohydrates were present. When subjected to chilling stress, mannitol levels dropped by 75%, sucrose by 52%, and inositol by 54% in the low-mannitol lines (M2 and M9). In M3 and M8, the high-mannitol lines, mannitol levels decreased by 36%, sucrose by 25%, and inositol by 56%, respectively. Raffinose increased 2- to 3-fold in all lines following exposure to low-temperature chilling stress. In the higher mannitol lines only 0.04% to 0.06% of the total osmotic potential generated from all solutes could be attributed to mannitol, thus its action is more like that of an osmoprotectant rather than an osmoregulator. This study demonstrates that metabolic engineering of osmoprotectant synthesis pathways can be used to improve stress tolerance in horticultural crops.

The potential clinical impact of pre-emptive screening of multiple polymorphisms in gene-encoding DPD on patients candidate for fluoropyrimidine based-chemotherapy: An experience of the Northern Italy Cancer Centre.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 2567-2567
Author(s):  
Francesco Iachetta ◽  
Angela Damato ◽  
Candida Bonelli ◽  
Alessandra Romagnani ◽  
Maria Banzi ◽  
...  
Keyword(s):  
Standard Dose ◽  
Dihydropyrimidine Dehydrogenase ◽  
The Other ◽  
Severe Toxicity ◽  
Wild Type ◽  
Gene Encoding ◽  
Mutational Status ◽  
Key Enzyme ◽  
Life Threatening ◽  
Potential Clinical Impact

2567 Background: Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the metabolism of fluorouracil. Deleterious polymorphisms in gene-encoding DPD ( DPYD) results in a DPD deficiency that causes life-threatening toxicities when the standard dose of fluorouracil is used. DPYD*2A (IVS14+1G > A) is the most common single-nucleotide polymorphism (SNP) associated with critical DPD deficiency. At present, most of the evidence supports screening for at least 3 SNPs (DPYD*2A, c.2846 A > T, c.1679T > G). The aim of this study is to confirm that the detection of additional polymorphisms of DPYD could enhance prevention of fluoropyrimidine toxicity. Methods: In 2011, we began to screen DPYD*2A in patients candidate for fluoropyrimidine based-chemotherapy. As the first step of the evaluation, we selected all cases of DPYD*2A wild type, from 2011 to 2012, who developed CTC-NCI-V.3 toxicity ≥ G3. In these patients, we researched the other 3 SNPs (c.2846 A > T, c.1679T > G, c.2194C > A). Mutational status was analyzed with real Time PCR. Results: From 2011 to 2016 we pre-emptively screened DPD deficiency in 1,863 patients and 32 subjects (1.6%), with results mutated for DPYD*2A. As the first step of the evaluation, 548 subjects were assessed from 2011 to 2012. We found 7 patients who were carriers of the DPYD*2A mutation (1.27%). Of the 541 wild type cases, 114 presented toxicities ≥ G3. In this subgroup, 22 patients (19%) proved to be mutated for the other SNPs of DPYP, as reported in the table below. Conclusions: Preliminary data show that in 22 (19%) of 114 patients who presented severe toxicity which was not correlated with DPYD*2A, we found other polymorphisms of gene encoding DPD. Out of the 3 SNPs evaluated, c.2194 C > A proved to be the most frequent, although it is the polymorphism that is least known and least studied. Such results suggest that the evaluation of additional polymorphisms could enhance the prevention of fluoropyrimidine toxicity. The results are expected to be clarified further in the second step, which is ongoing. [Table: see text]

scholarly journals Thiosulfate-Dependent Chemolithoautotrophic Growth of Bradyrhizobium japonicum

2010 ◽  
Vol 76 (8) ◽  
pp. 2402-2409 ◽  
Author(s):  
Sachiko Masuda ◽  
Shima Eda ◽  
Seishi Ikeda ◽  
Hisayuki Mitsui ◽  
Kiwamu Minamisawa
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Green Sulfur Bacteria ◽  
Growth Conditions ◽  
Deletion Mutation ◽  
Resting Cells ◽  
Wild Type ◽  
Sulfur Bacteria ◽  
Chemolithotrophic Growth ◽  
Green Sulfur ◽  
Sulfur Oxidizing

ABSTRACT Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B. japonicum USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the soxY 1 gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Alphaproteobacteria, left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY 2 gene at sox locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O2 respiration was observed only in USDA110 and the soxY 2 mutant and not in the soxY 1 mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O2. B. japonicum USDA110 showed 14CO2 fixation under chemolithotrophic growth conditions. The CO2 fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on sox locus I including the soxY 1 gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the Bradyrhizobiaceae, which phylogenetic analysis showed to be associated with the presence of sox locus I homologues, including the soxY 1 gene of B. japonicum USDA110.

scholarly journals The Impacts of Asparagus Extract Fractions on Growth and Fumonisins Biosynthesis in Fusarium Proliferatum

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 95 ◽  
Author(s):  
Witaszak ◽  
Lalak-Kańczugowska ◽  
Waśkiewicz ◽  
Stępień
Keyword(s):  
Fungal Biomass ◽  
Polyketide Synthase ◽  
Fumonisin B1 ◽  
Fold Increase ◽  
Dry Weight ◽  
Fusarium Proliferatum ◽  
Asparagus Officinalis ◽  
Future Research ◽  
Gene Encoding ◽  
Key Enzyme

Asparagus is a genus consisting of over two hundred species of perennial plants. Fusarium proliferatum is a major asparagus pathogen and it biosynthesizes a variety of mycotoxins, of which fumonisins B are prevalent. Our previous studies on F. proliferatum strains indicated that asparagus extract affects the expression of FUM1 gene, encoding polyketide synthase, a key enzyme of the FUM gene cluster governing the biosynthesis of fumonisins. An asparagus-derived F. proliferatum strain increased fumonisin B1 production after extract fractions’ addition, reaching the maximum 2 or 24 h after treatment. The cultures yielded between 40 and 520 mg of dry weight of mycelia after 14 days of cultivation. The differences in fungal biomass amounts between the whole extract and its fractions may result from synergistic effect of all bioactive compounds present in asparagus extract. Among extract fractions, the methanolic fraction had the highest effect on the dry weight of the mycelium reaching about a 13-fold increase compared to the control. Furthermore, we measured the relative expression of the FUM1 gene. Due to the possible antifungal activity of tested extract fractions, future research will be focused on the identification of the Asparagus officinalis L. compounds responsible for this activity.

scholarly journals Antioxidant Functions of Nitric Oxide Synthase in a Methicillin SensitiveStaphylococcus aureus

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Manisha Vaish ◽  
Vineet K. Singh
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Normal Growth ◽  
Growth Conditions ◽  
Nitric Oxide Donor ◽  
Wild Type ◽  
Gene Encoding ◽  
Stress Studies ◽  
Oxide Synthase

Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria includingStaphylococcus aureuspossess a gene encoding nitric oxide synthase (SaNOS) in their chromosome. In this study it was determined that under normal growth conditions, expression ofSaNOSwas highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion ofSaNOSled to increased susceptibility of the mutant cells compared to wild-typeS. aureus. While inhibition ofSaNOSactivity by the addition of L-NAME increased sensitivity of the wild-typeS. aureusto oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of theSaNOSmutant. TheSaNOSmutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-typeS. aureus.

scholarly journals Localization of the Bacillus subtilis murB Gene within the dcw Cluster Is Important for Growth and Sporulation

2006 ◽  
Vol 188 (5) ◽  
pp. 1721-1732 ◽  
Author(s):  
Gonçalo Real ◽  
Adriano O. Henriques
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Ectopic Expression ◽  
Normal Growth ◽  
Wild Type ◽  
Extra Copy ◽  
Gene Encoding ◽  
Key Enzyme ◽  
High Concentrations

ABSTRACT The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for “division and cell wall”) cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivIB levels were normal in a second mutant containing an in-frame deletion of murB (ΔmurB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the ΔmurB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a murB + PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.

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