scholarly journals Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

2015 ◽  
Vol 81 (12) ◽  
pp. 4098-4110 ◽  
Author(s):  
Binbin Sheng ◽  
Jing Xu ◽  
Yingxin Zhang ◽  
Tianyi Jiang ◽  
Sisi Deng ◽  
...  
Keyword(s):  
Energy Source ◽  
Carbon Source ◽  
Electron Acceptor ◽  
Gluconobacter Oxydans ◽  
The Novel ◽  
Hydroxy Acids ◽  
Lactate Oxidase ◽  
Content Type ◽  
Membrane Bound ◽  
Lactate Dehydrogenases

ABSTRACTd-Lactate was identified as one of the few available organic acids that supported the growth ofGluconobacter oxydans621H in this study. Interestingly, the strain usedd-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth ofG. oxydans621H ond-lactate was therefore investigated. Although two putative NAD-independentd-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizingd-lactate, GOX1253 was the only enzyme able to support thed-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity towardd-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity towardd-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously ind-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth ofG. oxydansond-lactate, but also provides new insights for applications of the important industrial microbe and the noveld-lactate oxidase.

scholarly journals Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

2006 ◽  
Vol 188 (21) ◽  
pp. 7668-7676 ◽  
Author(s):  
Tina Hölscher ◽  
Helmut Görisch
Keyword(s):  
Energy Source ◽  
Wild Type Strain ◽  
Gluconobacter Oxydans ◽  
Analysis Data ◽  
Pyrroloquinoline Quinone ◽  
Vital Role ◽  
Reverse Transcription Pcr ◽  
Membrane Bound ◽  
Pqq Biosynthesis

ABSTRACT In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membrane-bound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with d-mannitol, d-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with d-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQ-dependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.

scholarly journals Diversity and Ubiquity of Bacteria Capable of Utilizing Humic Substances as Electron Donors for Anaerobic Respiration

2002 ◽  
Vol 68 (5) ◽  
pp. 2445-2452 ◽  
Author(s):  
John D. Coates ◽  
Kimberly A. Cole ◽  
Romy Chakraborty ◽  
Susan M. O'Connor ◽  
Laurie A. Achenbach
Keyword(s):  
Energy Source ◽  
Carbon Source ◽  
Humic Substances ◽  
Electron Donor ◽  
Electron Acceptor ◽  
Electron Donors ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Couple Growth ◽  
Probable Number

ABSTRACT Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter, Geothrix, and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 × 101 in aquifer sediments to a high of 9.33 × 106 in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N2. Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the Proteobacteria. Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the Proteobacteria. The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

scholarly journals Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

2015 ◽  
Vol 81 (10) ◽  
pp. 3552-3560 ◽  
Author(s):  
Naoya Kataoka ◽  
Minenosuke Matsutani ◽  
Toshiharu Yakushi ◽  
Kazunobu Matsushita
Keyword(s):  
Mutant Strain ◽  
Gluconobacter Oxydans ◽  
Efficient Production ◽  
Broad Host Range ◽  
Putative Promoter Region ◽  
Content Type ◽  
Biotransformation Process ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Almost All

ABSTRACT2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate ford-tartrate and also vitamin C production. AlthoughGluconobacter oxydansNBRC3293 produces 2,5DKG fromd-glucose viad-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 residuald-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochromeccomplex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied thekgdSLCgenes encoding 2KGDH inG. oxydansNBRC3293 to improve 2,5DKG production byGluconobacterspp. ThekgdS,kgdL, andkgdCgenes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. ThekgdSLCgenes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene ofG. oxydansfor expression inGluconobacterspp. According to our results, 2KGDH that was purified from the recombinantGluconobactercells showed characteristics nearly the same as those reported previously. We also expressed thekgdSLCgenes in a mutant strain ofGluconobacter japonicusNBRC3271 (formerlyGluconobacter dioxyacetonicusIFO3271) engineered to produce 2KG efficiently from a mixture ofd-glucose andd-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose andd-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of aGluconobacterstrain that produces 2,5DKG efficiently and homogeneously.

scholarly journals Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

2013 ◽  
Vol 79 (9) ◽  
pp. 2882-2890 ◽  
Author(s):  
Guillermo D. Repizo ◽  
Víctor S. Blancato ◽  
Pablo Mortera ◽  
Juke S. Lolkema ◽  
Christian Magni
Keyword(s):  
Enterococcus Faecalis ◽  
Soluble Form ◽  
The Novel ◽  
Oxaloacetate Decarboxylase ◽  
Content Type ◽  
Membrane Pump ◽  
B Subunit ◽  
Single Deletion ◽  
Membrane Bound

ABSTRACTEnterococcus faecalisencodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits:E. faecaliscarboxyltransferase subunit OadA (termedEf-A), membrane pumpEf-B, biotin acceptor proteinEf-D, and the novel subunitEf-H. Our results show that inE. faecalis, subunitsEf-A,Ef-D, andEf-H form a cytoplasmic soluble complex (termedEf-AHD) which is also associated with the membrane. In order to characterize the role of the novelEf-H subunit, coexpression ofoadgenes was performed inEscherichia coli, showing that this subunit is vital forEf-A andEf-D interaction. Diminished growth of theoadAandoadDsingle deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, theoadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the solubleEf-AHD complex is functional inE. faecalis.These results suggest that theEf-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-boundEf-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Characterization of novel Gluconobacter species from fruits and fermented food products: Gluconobacter cadivus sp. nov., Gluconobacter vitians sp. nov. and Gluconobacter potus sp. nov

2021 ◽  
Author(s):  
Atena Sadat Sombolestani ◽  
Ilse Cleenwerck ◽  
Margo Cnockaert ◽  
Wim Borremans ◽  
Anneleen D. Wieme ◽  
...  
Keyword(s):  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
Gluconobacter Oxydans ◽  
Food Products ◽  
Fermented Food ◽  
Phylogenomic Analysis ◽  
The Novel ◽  
Content Type ◽  
Link Type

Strains LMG 1744T, LMG 1745, LMG 31484T, LMG 1764T and R-71646 were isolated from rotting fruits and fermented food products. A phylogenomic analysis based on 107 single-copy core genes revealed that they grouped in a Gluconobacter lineage comprising Gluconobacter oxydans , Gluconobacter roseus , Gluconobacter sphaericus, Gluconobacter kanchanaburiensis, Gluconobacter albidus, Gluconobacter cerevisiae, Gluconobacter kondonii and Gluconobacter aidae . OrthoANIu and digital DNA hybridization analyses demonstrated that these five strains represented three novel Gluconobacter species, which could be differentiated from the type strains of closely related Gluconobacter species by multiple phenotypic characteristics. We therefore propose to classify strains LMG 1744T and LMG 1745 in the novel species Gluconobacter cadivus sp. nov., with LMG 1744T (=CECT 30141T) as the type strain; to classify strain LMG 31484T as the novel species Gluconobacter vitians sp. nov., with LMG 31484T (=CECT 30132T) as the type strain; and to classify strains LMG 1764T and R-71646 in the novel species Gluconobacter potus sp. nov., with LMG 1764T (=CECT 30140T) as the type strain.

scholarly journals Dissimilatory Nitrate Reduction to Ammonium (DNRA) and Denitrification Pathways Are Leveraged by Cyclic AMP Receptor Protein (CRP) Paralogues Based on Electron Donor/Acceptor Limitation in Shewanella loihica PV-4

2020 ◽  
Vol 87 (2) ◽  
Author(s):  
Shuangyuan Liu ◽  
Jingcheng Dai ◽  
Hehong Wei ◽  
Shuyang Li ◽  
Pei Wang ◽  
...  
Keyword(s):  
Global Warming ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Electron Donor ◽  
Electron Acceptor ◽  
Nitrite Reductase ◽  
Nitrate Reduction ◽  
Receptor Protein ◽  
Content Type ◽  
Dissimilatory Nitrate Reduction

ABSTRACT Under anoxic conditions, many bacteria, including Shewanella loihica strain PV-4, could use nitrate as an electron acceptor for dissimilatory nitrate reduction to ammonium (DNRA) and/or denitrification. Previous and current studies have shown that DNRA is favored under higher ambient carbon-to-nitrogen (C/N) ratios, whereas denitrification is upregulated under lower C/N ratios, which is consistent with our bioenergetics calculations. Interestingly, computational analyses indicate that the common cyclic AMP receptor protein (designated CRP1) and its paralogue CRP2 might both be involved in the regulation of two competing dissimilatory nitrate reduction pathways, DNRA and denitrification, in S. loihica PV-4 and several other denitrifying Shewanella species. To explore the regulatory mechanism underlying the dissimilatory nitrate reduction (DNR) pathways, nitrate reduction of a series of in-frame deletion mutants was analyzed under different C/N ratios. Deletion of crp1 could accelerate the reduction of nitrite to NO under both low and high C/N ratios. CRP1 is not required for denitrification and actually suppresses production of NO and N2O gases. Deletion of either of the NO-forming nitrite reductase genes nirK or crp2 blocked production of NO gas. Furthermore, real-time PCR and electrophoretic mobility shift assays (EMSAs) demonstrated that the transcription levels of DNRA-relevant genes such as nap-β (napDABGH), nrfA, and cymA were upregulated by CRP1, while nirK transcription was dependent on CRP2. There are tradeoffs between the different physiological roles of nitrate/lactate, as nitrogen nutrient/carbon source and electron acceptor/donor and CRPs may leverage dissimilatory nitrate reduction pathways for maximizing energy yield and bacterial survival under ambient environmental conditions. IMPORTANCE Some microbes utilize different dissimilatory nitrate reduction (DNR) pathways, including DNR to ammonia (DNRA) and denitrification pathways, for anaerobic respiration in response to ambient carbon/nitrogen ratio changes. Large-scale industrial nitrogen fixation and fertilizer application raise the concern of emission of N2O, a stable gas with potent global warming potential, as consequence of microbial respiration, thereby aggravating global warming and climate change. However, little is known about the molecular mechanism underlying the choice of two competing DNR pathways. We demonstrate that the global regulator CRP1, which is widely encoded in bacteria, is required for DNRA in S. loihica PV-4 strain, while the CRP2 paralogue is required for transcription of the nitrite reductase gene nirK for denitrification. Sufficient carbon source lead to the predominance of DNRA, while carbon source/electron donor deficiency may result in an incomplete denitrification process, raising the concern of high levels of N2O emission from nitrate-rich and carbon source-poor waters and soils.

scholarly journals New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”

2014 ◽  
Vol 81 (4) ◽  
pp. 1338-1352 ◽  
Author(s):  
Kristina Lang ◽  
Jörg Schuldes ◽  
Andreas Klingl ◽  
Anja Poehlein ◽  
Rolf Daniel ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Physiological Data ◽  
Dual Function ◽  
The Novel ◽  
Heterodisulfide Reductase ◽  
Content Type ◽  
Methanosphaera Stadtmanae ◽  
Seventh Order ◽  
Membrane Bound ◽  
Energy Converting

ABSTRACTThe recently discovered seventh order of methanogens, theMethanomassiliicoccales(previously referred to as “Methanoplasmatales”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “CandidatusMethanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “Ca. Methanoplasma termitum” lacks the entire pathway for CO2reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates thatMethanomassiliicoccalesemploy a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophicMethanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F420:methanophenazine oxidoreductase (Fpo) ofMethanosarcinalesbut lacks an F420-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog inMethanosaeta thermophila. Since allMethanomassiliicoccalesalso lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide inMethanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.

Enrichment and characterization of an anaerobic methyl ethyl ketoxime degrading culture from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor

1998 ◽  
Vol 37 (4-5) ◽  
pp. 95-98 ◽  
Author(s):  
Nancy G. Love ◽  
Mary E. Rust ◽  
Kathy C. Terlesky
Keyword(s):  
Energy Source ◽  
Activated Sludge ◽  
Electron Acceptor ◽  
Sequencing Batch Reactor ◽  
Enrichment Culture ◽  
Batch Reactor ◽  
Nitrification Inhibitor ◽  
Time Frame ◽  
Methyl Ethyl

An anaerobic enrichment culture was developed from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor using methyl ethyl ketoxime (MEKO), a potent nitrification inhibitor, as the sole carbon and energy source in the absence of molecular oxygen and nitrate. The enrichment culture was gradually fed decreasing amounts of biogenic organic compounds and increasing concentrations of MEKO over 23 days until the cultures metabolized the oxime as the sole carbon source; the cultures were maintained for an additional 41 days on MEKO alone. Turbidity stabilized at approximately 100 mg/l total suspended solids. Growth on selective media plates confirmed that the microorganisms were utilizing the MEKO as the sole carbon and energy source. The time frame required for growth indicated that the kinetics for MEKO degradation are slow. A batch test indicated that dissolved organic carbon decreased at a rate comparable to MEKO consumption, while sulfate was not consumed. The nature of the electron acceptor in anaerobic MEKO metabolism is unclear, but it is hypothesized that the MEKO is hydrolyzed intracellularly to form methyl ethyl ketone and hydroxylamine which serve as electron donor and electron acceptor, respectively.

scholarly journals Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

2015 ◽  
Vol 81 (7) ◽  
pp. 2466-2473 ◽  
Author(s):  
Muhammad Farhan Ul-Haque ◽  
Bhagyalakshmi Kalidass ◽  
Alexey Vorobev ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Methane Monooxygenase ◽  
Particulate Methane Monooxygenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Methylosinus Trichosporium ◽  
Monooxygenase Activity ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
A Subunit ◽  
Membrane Bound

ABSTRACTMethanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced byMethylosinus trichosporiumOB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. WhenMethylosinus trichosporiumOB3b was grown in the presence of 1 μM CuCl2, expression ofmmoX, encoding a subunit of the hydroxylase component of sMMO, was very low.mmoXexpression increased, however, when methanobactin fromMethylocystissp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated withM. trichosporiumOB3b. IfM. trichosporiumOB3b was grown in the absence of CuCl2, themmoXexpression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure ofMethylosinus trichosporiumOB3b to SB2-Mb had no effect on expression ofmbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2.mbnAexpression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

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