Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

ABSTRACTIn this study, we focus on production of heterologous α-amylase in the yeastSaccharomyces cerevisiaeunder anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the α-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

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Evaluation of the rate of hydrolysis of slowly biodegradable COD (SBCOD) using starch as substrate under anaerobic, anoxic and aerobic conditions

Water Science & Technology ◽

10.2166/wst.1994.0559 ◽

1994 ◽

Vol 30 (11) ◽

pp. 191-199 ◽

Cited By ~ 22

Author(s):

Delfin C. San Pedro ◽

Takashi Mino ◽

Tomonori Matsuo

Keyword(s):

Activated Sludge ◽

Electron Acceptor ◽

Biomass Concentration ◽

Organic Substrate ◽

Hydrolysis Rate ◽

Anaerobic Conditions ◽

Final Electron ◽

Rate Of Hydrolysis ◽

Hydrolysis Of ◽

Final Electron Acceptor

In the Activated Sludge Model No. 1 proposed by an IAWPRC Task Group, the possible differences of the hydrolysis rate for the slowly biodegradable organic compounds (SBCOD) under aerobic, anoxic and anaerobic conditions are accounted for by the introduction of a correction factor ηh. In the present study, this factor ηh was experimentally evaluated using starch as organic substrate to represent SBCOD. It was found that after the activated sludge had been well acclimatized with starch, the hydrolysis rate is insignificantly affected by the condition of the final electron acceptor. The value of ηh approaches unity. Results also suggested that biomass concentration has no significant influence on hydrolysis rate of starch.

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Anaerobic Growth of Acidithiobacillus ferrooxidans on Pyrite

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.825.96 ◽

2013 ◽

Vol 825 ◽

pp. 96-99

Author(s):

Blanca Escobar ◽

Tomas Vargas

Keyword(s):

Electron Acceptor ◽

Bacterial Growth ◽

Ferrous Iron ◽

Elemental Sulfur ◽

Ferric Iron ◽

Anaerobic Respiration ◽

Anaerobic Growth ◽

Final Electron ◽

Anaerobic Reduction ◽

Final Electron Acceptor

In the bioleaching of mineral sulphides under the catalytic action ofAt. ferrooxidans,ferrous ion oxidation and sulfides/sulfur solubilization uses oxygen as the final electron acceptor. Also, under anaerobic conditions,At. ferrooxidanscan alternatively catalize the oxidation of sulfur or reduced inorganic sulfur compounds (RISC) using ferric iron as electron acceptor [1]. The formation of Fe (II) from pyrite and covellite in the ferric anaerobic bioleaching withA. ferrooxidans,has been studied and well documented [2,3]. The requirements of ferric iron as electron acceptor for the anaerobic growth ofAt. ferrooxidanson elemental sulfur has been demonstrated and a linear relationship was obtained between the concentration of ferrous iron accumulated in the cultures and the increase in cell density [4]. It has been suggested a possible role in the solubilization of metals from sulfide ores involving the participation of the enzyme sulfur (sulfide): Fe (III) oxidoreductase [5]. Bacterial growth ofAt. ferrooxidanshas also been reported in the oxidative anaerobic respiration using hydrogen as electron donor and ferric iron as electron acceptor [6]. Anaerobic reduction of ferric iron and ferrous iron production from pyrite byAt. ferrooxidanshas been demonstrated [2], however there are no reports about bacterial growth using this mineral. In this work, we studied the anaerobic bioleaching of pyrite with the aim to determine ifAt. ferrooxidansis capable to anaerobic growth on pyrite using ferric iron as electron acceptor.

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Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium ( Alphaproteobacteria : Rhodospirillaceae ) isolated from a salt marsh

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.044453-0 ◽

2013 ◽

Vol 63 (Pt_5) ◽

pp. 1824-1833 ◽

Cited By ~ 52

Author(s):

Dennis A. Bazylinski ◽

Timothy J. Williams ◽

Christopher T. Lefèvre ◽

Denis Trubitsyn ◽

Jiasong Fang ◽

...

Keyword(s):

Salt Marsh ◽

Electron Acceptor ◽

Anaerobic Growth ◽

Rrna Gene ◽

Magnetotactic Bacterium ◽

Single Chain ◽

Circular Chromosome ◽

Terminal Electron Acceptor ◽

Content Type ◽

Link Type

A magnetotactic bacterium, designated strain MV-1T, was isolated from sulfide-rich sediments in a salt marsh near Boston, MA, USA. Cells of strain MV-1T were Gram-negative, and vibrioid to helicoid in morphology. Cells were motile by means of a single polar flagellum. The cells appeared to display a transitional state between axial and polar magnetotaxis: cells swam in both directions, but generally had longer excursions in one direction than the other. Cells possessed a single chain of magnetosomes containing truncated hexaoctahedral crystals of magnetite, positioned along the long axis of the cell. Strain MV-1T was a microaerophile that was also capable of anaerobic growth on some nitrogen oxides. Salinities greater than 10 % seawater were required for growth. Strain MV-1T exhibited chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron acceptor (microaerobic growth) and on thiosulfate using nitrous oxide (N2O) as the terminal electron acceptor (anaerobic growth). Chemo-organoautotrophic and methylotrophic growth was supported by formate under microaerobic conditions. Autotrophic growth occurred via the Calvin–Benson–Bassham cycle. Chemo-organoheterotrophic growth was supported by various organic acids and amino acids, under microaerobic and anaerobic conditions. Optimal growth occurred at pH 7.0 and 26–28 °C. The genome of strain MV-1T consisted of a single, circular chromosome, about 3.7 Mb in size, with a G+C content of 52.9–53.5 mol%.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MV-1T belongs to the family Rhodospirillaceae within the Alphaproteobacteria , but is not closely related to the genus Magnetospirillum . The name Magnetovibrio blakemorei gen. nov., sp. nov. is proposed for strain MV-1T. The type strain of Magnetovibrio blakemorei is MV-1T ( = ATCC BAA-1436T = DSM 18854T).

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Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol

Applied and Environmental Microbiology ◽

10.1128/aem.00382-11 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4894-4904 ◽

Cited By ~ 79

Author(s):

Cong T. Trinh ◽

Johnny Li ◽

Harvey W. Blanch ◽

Douglas S. Clark

Keyword(s):

Escherichia Coli ◽

Anaerobic Growth ◽

Mode Analysis ◽

Glycolytic Flux ◽

Strictly Anaerobic ◽

Anaerobic Conditions ◽

Content Type ◽

E Coli ◽

Model Predictions ◽

Chromosomal Genes

ABSTRACTFermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design anEscherichia colistrain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism ofE. coliwas decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growthE. colicannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesignedE. colistrain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producingE. colistrain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.

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The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00120-17 ◽

2017 ◽

Vol 199 (16) ◽

Cited By ~ 9

Author(s):

Brian M. Meehan ◽

Cristina Landeta ◽

Dana Boyd ◽

Jonathan Beckwith

Keyword(s):

Escherichia Coli ◽

Disulfide Bond ◽

Bond Formation ◽

Anaerobic Growth ◽

Strictly Anaerobic ◽

Disulfide Bond Formation ◽

Anaerobic Conditions ◽

Content Type ◽

E Coli ◽

Laboratory Conditions

ABSTRACT Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited. IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

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Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02413-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7496-7508 ◽

Cited By ~ 23

Author(s):

Andrea Michel ◽

Abigail Koch-Koerfges ◽

Karin Krumbach ◽

Melanie Brocker ◽

Michael Bott

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Tricarboxylic Acid Cycle ◽

Model Organism ◽

Anaerobic Growth ◽

Efficient Production ◽

Ph Homeostasis ◽

Anaerobic Conditions ◽

Acid Fermentation ◽

Content Type

ABSTRACTCorynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions tol-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology ofC. glutamicumunder anaerobic conditions.

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Mitochondrial DNA Heteroplasmy in Candida glabrata after Mitochondrial Transformation

Eukaryotic Cell ◽

10.1128/ec.00349-09 ◽

2010 ◽

Vol 9 (5) ◽

pp. 806-814 ◽

Cited By ~ 17

Author(s):

Jingwen Zhou ◽

Liming Liu ◽

Jian Chen

Keyword(s):

Mitochondrial Dna ◽

Candida Glabrata ◽

Southern Blotting ◽

Selective Pressure ◽

Genetic Manipulation ◽

Direct Method ◽

Anaerobic Conditions ◽

Aerobic Conditions ◽

Content Type ◽

Mitochondrial Dna Heteroplasmy

ABSTRACT Genetic manipulation of mitochondrial DNA (mtDNA) is the most direct method for investigating mtDNA, but until now, this has been achieved only in the diploid yeast Saccharomyces cerevisiae. In this study, the ATP6 gene on mtDNA of the haploid yeast Candida glabrata (Torulopsis glabrata) was deleted by biolistic transformation of DNA fragments with a recoded ARG8 m mitochondrial genetic marker, flanked by homologous arms to the ATP6 gene. Transformants were identified by arginine prototrophy. However, in the transformants, the original mtDNA was not lost spontaneously, even under arginine selective pressure. Moreover, the mtDNA transformants selectively lost the transformed mtDNA under aerobic conditions. The mtDNA heteroplasmy in the transformants was characterized by PCR, quantitative PCR, and Southern blotting, showing that the heteroplasmy was relatively stable in the absence of arginine. Aerobic conditions facilitated the loss of the original mtDNA, and anaerobic conditions favored loss of the transformed mtDNA. Moreover, detailed investigations showed that increases in reactive oxygen species in mitochondria lacking ATP6, along with their equal cell division, played important roles in determining the dynamics of heteroplasmy. Based on our analysis of mtDNA heteroplasmy in C. glabrata, we were able to generate homoplasmic Δatp6 mtDNA strains.

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Anaerobic conditions unmask antimicrobial resistance

10.1101/2021.06.26.450051 ◽

2021 ◽

Author(s):

Ashton Creasy-Marrazzo ◽

Morteza M. Saber ◽

Manasi Kamat ◽

Laura S. Bailey ◽

Firdausi Qadri ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Association Studies ◽

Anaerobic Respiration ◽

Anaerobic Growth ◽

Genome Wide Association Studies ◽

Anaerobic Conditions ◽

Aerobic Conditions ◽

Minimal Inhibitory Concentrations ◽

Genome Wide ◽

The Impact

Antimicrobial resistance (AMR) in Gram negative enteropathogens is an urgent threat to the antibiotic formulary. These taxa undergo anaerobic respiration within the host, yet little is known about how anaerobic conditions influence antimicrobial resistance (AMR). The facultative enteropathogen Vibrio cholerae was chosen as a model to determine the impact of anaerobic growth on AMR because cholera is one of the few non-invasive diarrhoeal diseases for which antibiotics are indicated, albeit conditionally. V. cholerae isolates from a single outbreak were tested for resistance by minimal inhibitory concentrations (MIC) under aerobic and anaerobic conditions against clinically relevant antibiotics. Here we show that the odds of classifying isolates as resistant under anaerobic compared to aerobic conditions increased over 20 times for ciprofloxacin and 50 times for azithromycin, yet for doxycycline, all isolates remained below the breakpoint for resistance. Genome-wide association studies (GWAS) found significant associations between known and unknown genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations. In most cases, AMR phenotypes were more heritable, and more genes significantly associated with AMR were discovered, under anaerobic conditions compared to aerobic conditions. These findings challenge the paradigm of testing facultative enteropathogens for AMR under aerobic conditions alone. This experimental approach establishes a new, more sensitive framework to track and investigate mechanisms of AMR.

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Molecular Dissection of Bacterial Nanowires

mBio ◽

10.1128/mbio.00270-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 43

Author(s):

Thomas Boesen ◽

Lars Peter Nielsen

Keyword(s):

Conclusive Evidence ◽

Geobacter Sulfurreducens ◽

Electron Conduction ◽

Content Type ◽

Type Iv ◽

Pilin Subunit ◽

Type Iv Pilin ◽

Final Electron ◽

Conductive Properties ◽

Final Electron Acceptor

ABSTRACTThe discovery of bacterial conductive structures, termed nanowires, has intrigued scientists for almost a decade. Nanowires enable bacteria to transfer electrons over micrometer distances to extracellular electron acceptors such as insoluble metal oxides or electrodes. Nanowires are pilus based and inGeobacter sulfurreducensare composed of the type IV pilin subunit PilA. Multihemec-type cytochromes have been shown to attach to nanowire pili. Two hypotheses have been proposed for electron conduction in nanowires. The first (termed the metal-like conductivity or MLC hypothesis) claims that the pilus itself has the electron-conductive properties and the attached cytochromes mediate transfer to the final electron acceptor, whereas the second hypothesis (termed the superexchange conductivity or SEC hypothesis) suggests that electrons are “hopping” between heme groups in cytochromes closely aligned with the pilus as a scaffold. In their recent article inmBio, Vargas et al. [M. Vargas, N. S. Malvankar, P.-L. Tremblay, C. Leang, J. A. Smith, P. Patel, O. Snoeyenbos-West, K. P. Nevin, and D. R. Lovley, mBio 4(2):e00210-13, 2013] address this ambiguity through an analysis of strain Aro-5, aG. sulfurreducensPilA mutant lacking aromatic residues in the nonconserved portion of PilA. These residues were suspected of involvement in electron transport according to the MLC hypothesis. TheG. sulfurreducensmutant had reduced conductive properties, lending important support to the MLC hypothesis. The data also highlight the need for further and more conclusive evidence for one or the other hypothesis.

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Fosfomycin and Tobramycin in Combination Downregulate Nitrate Reductase GenesnarGandnarH, Resulting in Increased Activity against Pseudomonas aeruginosa under Anaerobic Conditions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00750-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5406-5414 ◽

Cited By ~ 15

Author(s):

Gerard McCaughey ◽

Deirdre F. Gilpin ◽

Thamarai Schneiders ◽

Lucas R. Hoffman ◽

Matt McKevitt ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Nitrate Reductase ◽

Growth Curves ◽

Anaerobic Growth ◽

Anaerobic Conditions ◽

Content Type ◽

Transposon Mutants ◽

Increased Activity ◽

Increased Susceptibility

ABSTRACTThe activity of aminoglycosides, which are used to treatPseudomonas aeruginosarespiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lungin vivo. In contrast, a 4:1 (wt/wt) combination of fosfomycin and tobramycin (F:T), which is under investigation for use in the treatment of CF lung infection, has increased activity againstP. aeruginosaunder anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests, including nitrate utilization assays, growth curves, and susceptibility testing. Notably, growth in subinhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (P< 0.05) nitrate reductase genesnarGandnarH, which are essential for normal anaerobic growth ofP. aeruginosa. Under anaerobic conditions, F:T significantly decreased (P< 0.001) nitrate utilization inP. aeruginosastrains PAO1, PA14, and PA14lasR::Gm, a mutant known to exhibit increased nitrate utilization. A similar effect was observed with two clinicalP. aeruginosaisolates. Growth curves indicate that nitrate reductase transposon mutants had reduced growth under anaerobic conditions, with these mutants also having increased susceptibility to F:T compared to the wild type under similar conditions. The results of this study suggest that downregulation of nitrate reductase genes resulting in reduced nitrate utilization is the mechanism underlying the increased activity of F:T under anaerobic conditions.

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