Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.
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ABSTRACT:
SUBMITTER: McIntire W
PROVIDER: S-EPMC1152798 | biostudies-other | 1985 Nov
REPOSITORIES: biostudies-other
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Project description:Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-delta-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.
| S-EPMC2075040 | biostudies-literature
Project description:Background2-keto-D-gluconic acid (2KGA) is widely used as a chemical intermediate in the cosmetic, pharmaceutical and environmental industries. Several microbial fermentation processes have been developed for production of 2KGA but these suffer from substrate/product inhibition, byproduct formation and low productivity. In previous work, we showed that 2KGA can be specifically produced from glucose (Glu) or gluconic acid (GA) by resting wild-type Gluconobacter oxydans DSM2003 cells, although substrate concentration was relatively low. In this study, we attempted to improve 2KGA productivity by G. oxydans DSM2003 by overexpressing the ga2dh gene, which encodes the membrane-bound gluconate-2-dehydrogenase enzyme (GA2DH).ResultsThe ga2dh gene was overexpressed in G. oxydans DSM2003 under the control of three promoters, P tufB , P ga2dh or P ghp0169 , respectively. Among the recombinant strains obtained, G. oxydans_tufB_ga2dh showed a similar growth rate to that of the control strain and displayed the highest specific productivity of 2KGA from GA, which was increased nearly twofold compared with that of the control strain during batch biotransformation. When biocatalysis conditions were optimized, with provision of sufficient oxygen during biotransformation, up to 480 g/L GA was completely utilized over 45 h by resting cells of G. oxydans_tufB_ga2dh and 453.3 g/L 2KGA was produced. A productivity of 10.07 g/L/h and a yield of 95.3 % were obtained. Overexpression of the ga2dh gene also significantly improved the conversion of Glu to 2KGA. Under optimized conditions, 270 g/L Glu was converted to 321 g/L 2KGA over 18 h, with a yield of 99.1 % and a productivity of 17.83 g/L/h. The glucose concentrations during the batch biotransformation and the 2KGA productivities achieved in this study were relatively high compared with the results of previous studies.ConclusionsThis study developed an efficient bacterial strain (G. oxydans_tufB_ga2dh) for the production of 2KGA by overexpressing the ga2dh gene in G. oxydans. Supply of sufficient oxygen enhanced the positive effect of gene overexpression on 2KGA production. Gluconobacter oxydans_tufB_ga2dh is thus a competitive species for use in 2KGA production.
| S-EPMC4939059 | biostudies-literature
Project description:NADPH-dependent 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-D-gluconate and D-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-D-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288?K. A crystal of the 5KGR-NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75?Å resolution. The crystal of the complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.6, c=62.9?Å. A crystal of the 5KGR-NADPH-5-keto-D-gluconate complex was prepared by soaking the 5KGR-NADPH complex crystal in reservoir solution supplemented with 100?mM 5-keto-D-gluconate and 10?mM NADPH for 20?min and diffracted X-rays to 2.26?Å resolution. The crystal of the ternary complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.7, c=62.5?Å. Both crystals contained two molecules in the asymmetric unit.
| S-EPMC2998383 | biostudies-literature
Project description:We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37 degrees C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37 degrees C. The amount of ketogluconates produced at 37 degrees C was slightly less than the amount produced at 30 degrees C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30 degrees C and 50% at 37 degrees C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl(2) in vitro when cells were grown at 37 degrees C. Addition of 5 mM CaCl(2) to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.
| S-EPMC2704797 | biostudies-literature
Project description:High-throughput screening is a powerful tool for discovering strains in the natural environment that may be suitable for target production. Herein, a novel enzyme-based high-throughput screening method was developed for rapid screening of strains overproducing 2-keto-L-gulonic acid (2-KLG). The screening method detects changes in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm using a microplate reader when 2-KLG is degraded by 2-KLG reductase. In this research, three different 2-KLG reductases were expressed, purified, and studied. The 2-KLG reductase from Aspergillus niger were selected as the best appropriate reductase to establishment the method for its high activity below pH 7. Using the established method, and coupled with fluorescence-activated cell sorting, we achieved a high 2-KLG-producing strain of Gluconobacter oxydans WSH-004 from soil. When cultured with D-sorbitol as the substrate, the 2-KLG yield was 2.5 g/L from 50 g/L D-sorbitol without any side products. Compared with other reported screening methods, our enzyme-based method is more efficient and accurate for obtaining high-producing 2-KLG strains, and it is also convenient and cost-effective. The method is broadly applicable for screening keto acids and other products that can be oxidized via nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+).
| S-EPMC6923176 | biostudies-literature
Project description:2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.
| S-EPMC4407217 | biostudies-literature
Project description:The objective of the current study was to understand the glutaraldehyde resistance mechanisms in P. fluorescens and P. aeruginosa biofilms. Glutaraldehyde is a common biocide used in various industries to control the microbial growth. Recent reports of emergence of glutaraldehyde resistance in several bacterial species motivated this study to understand the genetic factors responsible got glutaraldehyde resistance. Using a combination of phenotypic assays, chemical genetic assays and RNA-seq, we demonstrate that novel efflux pump, polyamine biosynthesis, lipid biosynthesis and phosphonate degradation play significant role in glutaraldehyde resistance and post-glutaraldehyde recovery of Psudomonad biofilms.
2015-04-06 | GSE64448 | GEO
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen with a complex respiratory chain. The bacterium is predicted to express three NADH:ubiquinone oxidoreductases (NDH-1, NDH-2 and Nqr). We created deletions strains of the predicted NADH:ubiquinone oxidoreductases alone, and in combination to determine the respective roles of the NADH dehydrogenases in growth and virulence. NDH-1 and NDH-2 were largely redundant under aerobic conditions. Aerobic NADH dehydrogenase enzymatic activity assay was lost with deletion of both NDH-1 and NDH-2. Under anaerobic conditions, NDH-1 was required for robust growth, and overexpression of NDH-2 rescued the NDH-1 anaerobic growth defect in rich media. There was not compensatory upregulation of NDH-2 under anaerobic conditions in NDH-1 deletion strains. To test which genes were required for in vivo virulence, we used both an insect and plant disease model. In the Galleria mellonella model, neither deletion of NDH-1 nor NDH-2 led to a change in median lethal dose, although death occurred more slowly in the NDH-1 deletion infections. In a lettuce model of virulence, loss of NDH-1 caused a decrease in recovered viable bacteria and a decrease in visual tissue damage. The compound deletion of NDH-1/NDH-2 causes a severe growth defect, both under aerobic and anaerobic conditions, and was avirulent in a lettuce model. Together, these results demonstrate the redundancy of the P. aeruginosa respiratory chain at the NADH dehydrogenase level in aerobic growth and virulence.
| S-EPMC6370648 | biostudies-literature
Project description:Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD(+)- or NADP(+)-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological roles of the ALDH family remain unknown. The PAO1 strain upregulated HDH in the presence of the hydrazone adipic acid bis(ethylidene hydrazide) (AEH). Gene disruption of the HDH-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double gene disruption of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of group X ALDHs from Escherichia coli, Paracoccus denitrificans, and Ochrobactrum anthropi also acted as HDHs in that they produced HDH activity in the cells and degraded hydrazones. These findings indicated the physiological roles of group X ALDHs in bacteria and showed that they comprise a distinct ALDH subfamily.
| S-EPMC3294850 | biostudies-literature
Project description:A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms.
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