scholarly journals Physiological roles of short-chain and long-chain menaquinones (vitamin K2) in Lactococcus cremoris

2021 ◽  
Author(s):  
Yue Liu ◽  
Nikolaos Charamis ◽  
Sjef Boeren ◽  
Joost Blok ◽  
Alisha Geraldine Lewis ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Bacterial Species ◽  
Biomass Accumulation ◽  
Vitamin K2 ◽  
Physiological Significance ◽  
Short Chain ◽  
Extracellular Electron Transfer ◽  
Growth And Survival ◽  
Genes Encoding ◽  
Long Chain

Lactococcus cremoris and L. lactis are well-known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable amount of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer (ΔmenF), (ii) a presumed MK-1 producer (ΔispA) and (iii) a MK-3 producer (Δllmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.

scholarly journals Syntrophic interspecies electron transfer drives carbon fixation and growth by Rhodopseudomonas palustris under dark, anoxic conditions

2021 ◽  
Vol 7 (27) ◽  
pp. eabh1852
Author(s):  
Xing Liu ◽  
Lingyan Huang ◽  
Christopher Rensing ◽  
Jie Ye ◽  
Kenneth H. Nealson ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Carbon Fixation ◽  
Rhodopseudomonas Palustris ◽  
Reducing Power ◽  
Extracellular Electron Transfer ◽  
Geobacter Metallireducens ◽  
Electron Transfer Pathway ◽  
Anoxygenic Phototrophs ◽  
Anoxic Environments ◽  
Genes Encoding

In natural anoxic environments, anoxygenic photosynthetic bacteria fix CO2 by photoheterotrophy, photoautotrophy, or syntrophic anaerobic photosynthesis. Here, we describe electroautotrophy, a previously unidentified dark CO2 fixation mode enabled by the electrosyntrophic interaction between Geobacter metallireducens and Rhodopseudomonas palustris. After an electrosyntrophic coculture is formed, electrons are transferred either directly or indirectly (via electron shuttles) from G. metallireducens to R. palustris, thereby providing reducing power and energy for the dark CO2 fixation. Transcriptomic analyses demonstrated the high expression of genes encoding for the extracellular electron transfer pathway in G. metallireducens and the Calvin-Benson-Bassham carbon fixation cycle in R. palustris. Given that sediments constitute one of the most ubiquitous and abundant niches on Earth and that, at depth, most of the sedimentary niche is both anoxic and dark, dark carbon fixation provides a metabolic window for the survival of anoxygenic phototrophs, as well as an as-yet unappreciated contribution to the global carbon cycle.

scholarly journals Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates

2019 ◽  
Author(s):  
Christopher M. Dundas ◽  
Benjamin K. Keitz
Keyword(s):  
Electron Transfer ◽  
Dynamic Range ◽  
Reduction Rate ◽  
Shewanella Oneidensis ◽  
Logic Gates ◽  
Extracellular Electron Transfer ◽  
Response Curves ◽  
Fluorescent Reporters ◽  
Genes Encoding ◽  
Functional Understanding

AbstractExtracellular electron transfer pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging since a functional understanding of its EET pathway proteins and their effect on engineering parameterizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures, that predictably turn on and turn off EET flux, respectively, in response to IPTG. Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs. inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

scholarly journals Disentangling the syntrophic electron transfer mechanisms of Candidatus geobacter eutrophica through electrochemical stimulation and machine learning

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heyang Yuan ◽  
Xuehao Wang ◽  
Tzu-Yu Lin ◽  
Jinha Kim ◽  
Wen-Tso Liu
Keyword(s):  
Machine Learning ◽  
Electron Transfer ◽  
Hydrogen Transfer ◽  
Draft Genome ◽  
Extracellular Electron Transfer ◽  
Hydrogen Metabolism ◽  
Genetic Potential ◽  
Fermentative Bacteria ◽  
Genes Encoding ◽  
Transfer Mechanisms

AbstractInterspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation, indicating Ca. G. eutrophica’s EET ability. The high-quality draft genome further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. A Bayesian network was trained with the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis from dominant fermentative bacteria, Geobacter, and Methanobacterium. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

scholarly journals Disentangling Syntrophic Electron Transfer Mechanisms in Methanogenesis Through Electrochemical Stimulation, Omics, and Machine Learning

2021 ◽  
Author(s):  
Heyang Yuan ◽  
Xuehao Wang ◽  
Tzu-Yu Lin ◽  
Jinha Kim ◽  
Wen-Tso Liu
Keyword(s):  
Machine Learning ◽  
Electron Transfer ◽  
Bayesian Network ◽  
Methane Production ◽  
Extracellular Electron Transfer ◽  
Hydrogen Metabolism ◽  
Electrochemical Reactors ◽  
The Core ◽  
Modeling Approach ◽  
Genes Encoding

Abstract Background: Interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. Results: To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, acetate accumulated in all reactors, while propionate was detected only in the electrochemical reactors. Four dominant fermentative bacteria were identified in the core population, and metatranscriptomics analysis suggested that they were responsible for the degradation of fructose and ethanol to propionate, propanol, acetate, and H2. Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation. The results thus confirmed Ca. G. eutrophica’s EET ability. The high-quality draft genome (completeness 99.4%, contamination 0.6%) further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. Redundancy analysis and a Bayesian network constructed with the core population predicted the importance of Ca. G. eutrophica-related OTU650 to methane production. The Bayesian network modeling approach was also applied to the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis.Conclusions: Ca. G. eutrophica is electroactive and simultaneously performs IHT and DIET. The results from the metatranscriptomic analysis have provided valuable information for enrichment and isolation of Ca. G. eutrophica. IHT is predicted to have a stronger impact on methane production than DIET in the electrochemical reactors. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

scholarly journals Phylogenetic and structural diversity of aromatically dense pili from environmental metagenomes

2019 ◽  
Author(s):  
M. S. Bray ◽  
J. Wu ◽  
C.C. Padilla ◽  
F. J. Stewart ◽  
D. A. Fowle ◽  
...  
Keyword(s):  
Amino Acids ◽  
Electron Transfer ◽  
Microbial Respiration ◽  
Aromatic Amino Acids ◽  
Structural Diversity ◽  
Type Iv Pili ◽  
Extracellular Electron Transfer ◽  
Environmental Distribution ◽  
Type Iv ◽  
Genes Encoding

SummaryElectroactive type IV pili, or e-pili, are used by some microbial species for extracellular electron transfer. Recent studies suggest that e-pili may be more phylogenetically and structurally diverse than previously assumed. Here, we used updated aromatic density thresholds (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps, and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57) to search for putative e-pilin genes in metagenomes from diverse ecosystems with active microbial metal cycling. Environmental putative e-pilins were diverse in length and phylogeny, and included truncated e-pilins inGeobacterspp., as well as longer putative e-pilins in Fe(II)-oxidizingBetaproteobacteriaandZetaproteobacteria.Originality and SignificanceElectroactive pili (e-pili) are used by microorganisms to respire solid metals in their environment through extracellular electron transfer. Thus, e-pili enable microbes to occupy specific environmental niches. Additionally, e-pili have important potential for biotechnological applications. Currently the repertoire of known e-pili is small, and their environmental distribution is largely unknown. Using sequence analysis, we identified numerous genes encoding putative e-pili from diverse anoxic, metal-rich ecosystems. Our results expand the diversity of putative e-pili in environments where metal oxides may be important electron acceptors for microbial respiration.

scholarly journals Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

2016 ◽  
Vol 82 (17) ◽  
pp. 5428-5443 ◽  
Author(s):  
Sarah E. Barchinger ◽  
Sahand Pirbadian ◽  
Christine Sambles ◽  
Carol S. Baker ◽  
Kar Man Leung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Shewanella Oneidensis ◽  
Oxygen Limitation ◽  
Extracellular Electron Transfer ◽  
Transport Pathways ◽  
Content Type ◽  
Genes Encoding

ABSTRACTIn limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacteriumShewanella oneidensisMR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates thatS. oneidensisMR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of themtrAandmtrChomologsmtrFandmtrDeither remains unaffected or decreases under these conditions. TheompWgene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream ofmtrCandomcA. The transcriptome and mutant analyses ofS. oneidensisMR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires.IMPORTANCEShewanella oneidensisMR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain whyS. oneidensiscan be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer.Shewanellacoordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Substrate use prioritization by a coculture of five species of gut bacteria fed mixtures of arabinoxylan, xyloglucan, β-glucan, and pectin

2020 ◽  
Author(s):  
Y Liu ◽  
AL Heath ◽  
B Galland ◽  
N Rehrer ◽  
L Drummond ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Dietary Fiber ◽  
Plant Cell Wall ◽  
Bacterial Species ◽  
Short Chain ◽  
Emergent Properties ◽  
Human Gut ◽  
Fermentation Products ◽  
Plant Polysaccharides ◽  
Human Gut Microbiota

© 2020 American Society for Microbiology. Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, β-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula. Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology. This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

scholarly journals β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization

2020 ◽  
Vol 9 (1) ◽  
pp. 55
Author(s):  
María Florencia Eberhardt ◽  
José Matías Irazoqui ◽  
Ariel Fernando Amadio
Keyword(s):  
Bacterial Species ◽  
Value Added ◽  
Raw Material ◽  
Activity Levels ◽  
Treatment Technology ◽  
Catalytic Domains ◽  
Stabilization Ponds ◽  
Cazy Database ◽  
Genes Encoding ◽  
Value Added Products

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. β-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding β-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with β-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-β-galactoside and lactose. The activity levels of one of these novel β-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic β-galactosidases from diary stabilization ponds’ metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries’ by-products.

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