Combining metabolic flux analysis with proteomics to shed light on the metabolic flexibility: the case of Desulfovibrio vulgaris Hildenborough.

Introduction: Desulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments" it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions. Methods: To lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis). Results: The iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism. Discussion: In particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility.

Desulfovibrio desulfuricans and its derived metabolites confer resistance to FOLFOX through METTL3.

BACKGROUND: Chemoresistance is a critical factor contributing to poor prognosis in clinical patients with cancer undergoing postoperative adjuvant chemotherapy. The role of gut microbiota in mediating resistance to tumour chemotherapy remains to be investigated. METHODS: Patients with CRC were categorised into clinical benefit responders (CBR) and no clinical benefit responders (NCB) based on chemotherapy efficacy. Differential bacterial analysis using 16S rRNA sequencing revealed Desulfovibrio as a distinct microbe between the two groups. Employing a syngeneic transplantation model, we assessed the effect of Desulfovibrio on chemotherapy by measuring tumour burden, weight, and Ki-67 expression. We further explored the mechanisms underlying the compromised chemotherapeutic efficacy of Desulfovibrio using metabolomics, western blotting, colony formation, and cell apoptosis assays. FINDINGS: In comparison, Desulfovibrio was more abundant in the NCB group. In vivo experiments revealed that Desulfovibrio colonisation in the gut weakened the efficacy of FOLFOX. Treatment with Desulfovibrio desulfuricans elevates serum S-adenosylmethionine (SAM) levels. Interestingly, SAM reduced the sensitivity of CRC cells to FOLFOX, thereby promoting the growth of CRC tumours. These experiments suggest that SAM promotes the growth and metastasis of CRC by driving the expression of methyltransferase-like 3 (METTL3). INTERPRETATION: A high abundance of Desulfovibrio in the intestines indicates poor therapeutic outcomes for postoperative neoadjuvant FOLFOX chemotherapy in CRC. Desulfovibrio drives the manifestation of METTL3 in CRC, promoting resistance to FOLFOX chemotherapy by increasing the concentration of SAM. FUNDING: This study is supported by Wuxi City Social Development Science and Technology Demonstration Project (N20201005).

Hydrogen Sulfide Producers Drive a Diarrhea-Like Phenotype and a Methane Producer Drives a Constipation-Like Phenotype in Animal Models.

BACKGROUND: We recently demonstrated that diarrhea-predominant irritable bowel syndrome (IBS-D) subjects have higher relative abundance (RA) of hydrogen sulfide (H2S)-producing Fusobacterium and Desulfovibrio species, and constipation-predominant IBS (IBS-C) subjects have higher RA of methanogen Methanobrevibacter smithii. AIMS: In this study, we investigate the effects of increased methanogens or H2S producers on stool phenotypes in rat models. METHODS: Adult Sprague-Dawley rats were fed high-fat diet (HFD) for 60 days to increase M. smithii levels, then gavaged for 10 days with water (controls) or methanogenesis inhibitors. To increase H2S producers, rats were gavaged with F. varium or D. piger. Stool consistency (stool wet weight (SWW)) and gas production were measured. 16S rRNA gene sequencing was performed on stool samples. RESULTS: In HFD diet-fed rats (N = 30), stool M. smithii levels were increased (P < 0.001) after 52 days, correlating with significantly decreased SWW (P < 0.0001) at 59 days (R = - 0.38, P = 0.037). Small bowel M. smithii levels decreased significantly in lovastatin lactone-treated rats (P < 0.0006), and SWW increased (normalized) in lovastatin hydroxyacid-treated rats (P = 0.0246), vs. controls (N = 10/group). SWW increased significantly in D. piger-gavaged rats (N = 16) on day 10 (P < 0.0001), and in F. varium-gavaged rats (N = 16) at all timepoints, vs. controls, with increased stool H2S production. 16S sequencing revealed stool microbiota alterations in rats gavaged with H2S producers, with higher relative abundance (RA) of other H2S producers, particularly Lachnospiraceae and Bilophila in F. varium-gavaged rats, and Sutterella in D. piger-gavaged rats. CONCLUSIONS: These findings suggest that increased M. smithii levels result in a constipation-like phenotype in a rat model that is partly reversible with methanogenesis inhibitors, whereas gavage with H2S producers D. piger or F. varium results in increased colonization with other H2S producers and diarrhea-like phenotypes. This supports roles for the increased RA of methanogens and H2S producers identified in IBS-C and IBS-D subjects, respectively, in contributing to stool phenotypes.

Fecal microbiota as a predictor of acupuncture responses in patients with postpartum depressive disorder.

Background: There are several clinical and molecular predictors of responses to antidepressant therapy. However, these markers are either too subjective or complex for clinical use. The gut microbiota could provide an easily accessible set of biomarkers to predict therapeutic efficacy, but its value in predicting therapy responses to acupuncture in patients with depression is unknown. Here we analyzed the predictive value of the gut microbiota in patients with postpartum depressive disorder (PPD) treated with acupuncture. Methods: Seventy-nine PPD patients were enrolled: 55 were treated with acupuncture and 24 did not received any treatment. The 17-item Hamilton depression rating scale (HAMD-17) was used to assess patients at baseline and after eight weeks. Patients receiving acupuncture treatment were divided into an acupuncture-responsive group or non-responsive group according to HAMD-17 scores changes. Baseline fecal samples were obtained from the patients receiving acupuncture and were analyzed by high-throughput 16S ribosomal RNA sequencing to characterize the gut microbiome. Results: 47.27% patients responded to acupuncture treatment and 12.5% patients with no treatment recovered after 8-week follow-up. There was no significant difference in α-diversity between responders and non-responders. The ß-diversity of non-responders was significantly higher than responders. Paraprevotella and Desulfovibrio spp. were significantly enriched in acupuncture responders, and these organisms had an area under the curve of 0.76 and 0.66 for predicting responder patients, respectively. Conclusions: Paraprevotella and Desulfovibrioare may be useful predictive biomarkers to predict PPD patients likely to respond to acupuncture. Larger studies and validation in independent cohorts are now needed to validate our findings.

Biomineralization of Sulfate-Reducing Bacteria In Situ-Induced Preparation of Nano Fe2 O3 -Fe(Ni)S/C as High-Efficiency Oxygen Evolution Electrocatalyst.

Transition metal-based catalysts possess high catalytic activity for oxygen evolution reaction (OER). However, the preparation of high-performance OER electrocatalysts using simple strategies with a low cost still faces a major challenge. Herein, this work presents an innovative, in situ-induced preparation of the Fe2 O3 , FeS, and NiS nanoparticles, supported on carbon blacks (CBs) (denoted as Fe2 O3 -Fe(Ni)S/C) as a high-efficiency oxygen evolution electrocatalyst by employing biomineralization. Biomineralization, a simple synthesis strategy, demonstrates a huge advantage in controlling the size of the Fe2 O3 and Fe(Ni)S nanoparticles, as well as achieving uniform nanoparticle distribution on carbon blacks. It is found that the electrocatalyst Fe2 O3 -Fe(Ni)S/C-200 shows a good OER electrocatalytic activity with a small loading capacity, and it has a small overpotential and Tafel slope in 1 m KOH solution with values of 264 mV and 42 mV dec-1 , respectively, at a current density of 10 mA cm-2 . Additionally, it presents good electrochemical stability for over 24 h. The remarkable and robust electrocatalytic performance of Fe2 O3 -Fe(Ni)S/C-200 is attributed to the synergistic effect of Fe2 O3 , FeS, and doped-Ni species as well as its distinct 3D spherical structure. This approach indicates the promising applications of biomineralization for the bio-preparation of functional materials and energy conversion.

Microorganisms for the oxidation of nitrated cellulose in its effluents (review).

The processes of microbiological destruction of toxic and large-tonnage waste are the most attractive processes for protecting the environment. The review considers the results of studies of microbial decomposition of nitrate esters, including hardly decomposable nitrocellulose. The published data show that specific microorganisms are able to degrade nitrated cellulose compounds under both anaerobic and aerobic conditions. The most promising microorganisms in terms of the efficiency of the nitrocellulose degradation process are bacteria belonging to Desulfovibrio genera, fungi Fusarium solani and Sclerotium rolfsii, as well as their co-cultivation. Recently, the first information about the enzymes involved in the process of nitrocellulose degradation, possible mechanisms of reactions carried out by these enzymes, and the effect of electron donors and acceptors adding to the process have been obtained. Contamination of industrial wastewater with nitrocellulose leads to treatment necessity by using cost-effective, harmless methods. A combined aerobic-anaerobic system, including both bacteria and fungi, has shown hopeful results.

Hyperimmune bovine colostrum containing lipopolysaccharide antibodies (IMM124-E) has a nondetrimental effect on gut microbial communities in unchallenged mice.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea with the potential to cause long-term gastrointestinal (GI) dysfunction. Preventative treatments for ETEC-induced diarrhea exist, yet the effects of these treatments on GI commensals in healthy individuals are unclear. Whether administration of a prophylactic preventative treatment for ETEC-induced diarrhea causes specific shifts in gut microbial populations in controlled environments is also unknown. Here, we studied the effects of a hyperimmune bovine colostrum (IMM-124E) used in the manufacture of Travelan (AUST L 106709) on GI bacteria in healthy C57BL/6 mice. Using next-generation sequencing, we aimed to test the onset and magnitude of potential changes to the mouse gut microbiome in response to the antidiarrheagenic hyperimmune bovine colostrum product, rich in immunoglobulins against select ETEC strains (Travelan, Immuron Ltd). We show that in mice administered colostrum containing lipopolysaccharide (LPS) antibodies, there was an increased abundance of potentially gut-beneficial bacteria, such as Akkermansia and Desulfovibrio, without disrupting the underlying ecology of the GI tract. Compared to controls, there was no difference in overall weight gain, body or cecal weights, or small intestine length following LPS antibody colostrum supplementation. Overall, dietary supplementation with colostrum containing LPS antibodies produced subtle alterations in the gut bacterial composition of mice. Primarily, Travelan LPS antibody treatment decreased the ratio of Firmicutes/Bacteroidetes in gut microbial populations in unchallenged healthy mice. Further studies are required to examine the effect of Travelan LPS antibody treatment to engineer the microbiome in a diseased state and during recovery.

Biomineralization of uranium by Desulfovibrio desulfuricans A3-21ZLL under various hydrochemical conditions.

Uranium pollution in groundwater environment has become an important issue of global concern. In this study, a strain of Desulfovibrio desulfuricans was isolated from the tailings of acid heap leaching, and was shown to be able to remove uranium from water via biosorption, bio-reduction, passive biomineralization under uranium stress, and active metabolically dependent bioaccumulation. This research explored the effects of nutrients, pH, initial uranium and sulfate concentration on the functional groups, uranium valence, and crystal size and morphology of uranium immobilization products. Results showed that tetravalent and hexavalent phosphorus-containing uranium minerals was both formed. In sulfate-containing water where Desulfovibrio desulfuricans A3-21ZLL can grow, the sequestration of uranium by bio-reduction was significantly enhanced compared to that with no sulfate loading or no growth. Ungrown Desulfovibrio desulfuricans A3-21ZLL or dead ones released inorganic phosphate group in response to the stress of uranium, which associated with soluble uranyl ion to form insoluble uranium-containing precipitates. This study revealed the influence of hydrochemical conditions on the mineralogy characteristics and spatial distribution of microbial uranium immobilization products. This study is conducive to the long-term and stable bioremediation of groundwater in decommissioned uranium mining area.

Syntrophic Interactions Ameliorate Arsenic Inhibition of Solvent-Dechlorinating Dehalococcoides mccartyi.

Interactions and nutrient exchanges among members of microbial communities are important for understanding functional relationships in environmental microbiology. We can begin to elucidate the nature of these complex systems by taking a bottom-up approach utilizing simplified, but representative, community members. Here, we assess the effects of a toxic stress event, the addition of arsenite (As(III)), on a syntrophic co-culture containing lactate-fermenting Desulfovibrio vulgaris Hildenborough and solvent-dechlorinating Dehalococcoides mccartyi strain 195. Arsenic and trichloroethene (TCE) are two highly prevalent groundwater contaminants in the United States, and the presence of bioavailable arsenic is of particular concern at remediation sites in which reductive dechlorination has been employed. While we previously showed that low concentrations of arsenite (As(III)) inhibit the keystone TCE-reducing microorganism, D. mccartyi, this study reports the utilization of physiological analysis, transcriptomics, and metabolomics to assess the effects of arsenic on the metabolisms, gene expression, and nutrient exchanges in the described co-culture. It was found that the presence of D. vulgaris ameliorated arsenic stress on D. mccartyi, improving TCE dechlorination under arsenic-contaminated conditions. Nutrient and amino acid export by D. vulgaris may be a stress-ameliorating exchange in this syntrophic co-culture under arsenic stress, based on upregulation of transporters and increased extracellular nutrients like sarcosine and ornithine. These results broaden our knowledge of microbial community interactions and will support the further development and implementation of robust bioremediation strategies at multi-contaminant sites.

A Comparative Multi-Frequency EPR Study of Dipolar Interaction in Tetra-Heme Cytochromes.

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer.

Antibiofilm assay for antimicrobial peptides combating the sulfate-reducing bacteria Desulfovibrio vulgaris.

In medical, environmental, and industrial processes, the accumulation of bacteria in biofilms can disrupt many processes. Antimicrobial peptides (AMPs) are receiving increasing attention in the development of new substances to avoid or reduce biofilm formation. There is a lack of parallel testing of the effect against biofilms in this area, as well as in the testing of other antibiofilm agents. In this paper, a high-throughput screening was developed for the analysis of the antibiofilm activity of AMPs, differentiated into inhibition and removal of a biofilm. The sulfate-reducing bacterium Desulfovibrio vulgaris was used as a model organism. D. vulgaris represents an undesirable bacterium, which is considered one of the major triggers of microbiologically influenced corrosion. The application of a 96-well plate and steel rivets as a growth surface realizes real-life conditions and at the same time establishes a flexible, simple, fast, and cost-effective assay. All peptides tested in this study demonstrated antibiofilm activity, although these peptides should be individually selected depending on the addressed aim. For biofilm inhibition, the peptide DASamP1 is the most suitable, with a sustained effect for up to 21 days. The preferred peptides for biofilm removal are S6L3-33, in regard to bacteria reduction, and Bactenecin, regarding total biomass reduction.

Targeting Desulfovibrio vulgaris flagellin-induced NAIP/NLRC4 inflammasome activation in macrophages attenuates ulcerative colitis.

INTRODUCTION: The perturbations of gut microbiota could interact with excessively activated immune responses and play key roles in the etiopathogenesis of ulcerative colitis (UC). Desulfovibrio, the most predominant sulfate reducing bacteria (SRB) resided in the human gut, was observed to overgrow in patients with UC. The interactions between specific gut microbiota and drugs and their impacts on UC treatment have not been demonstrated well. OBJECTIVES: This study aimed to elucidate whether Desulfovibrio vulgaris (D. vulgaris, DSV) and its flagellin could activate nucleotide-binding oligomerization domain-like receptors (NLR) family of apoptosis inhibitory proteins (NAIP) / NLR family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome and promote colitis, and further evaluate the efficacy of eugeniin targeting the interaction interface of D. vulgaris flagellin (DVF) and NAIP to attenuate UC. METHODS: The abundance of DSV and the occurrence of macrophage pyroptosis in human UC tissues were investigated. Colitis in mice was established by dextran sulfate sodium (DSS) and gavaged with DSV or its purified flagellin. NAIP/NLRC4 inflammasome activation and macrophage pyroptosis were evaluated in vivo and in vitro. The effects of eugeniin on blocking the interaction of DVF and NAIP/NLRC4 and relieving colitis were also assessed. RESULTS: The abundance of DSV increased in the feces of patients with UC and was found to be associated with disease activity. DSV and its flagellin facilitated DSS-induced colitis in mice. Mechanistically, RNA sequencing showed that gene expression associated with inflammasome complex and pyroptosis was upregulated after DVF treatment in macrophages. DVF was further demonstrated to induce significant macrophage pyroptosis in vitro, depending on NAIP/NLRC4 inflammasome activation. Furthermore, eugeniin was screened as an inhibitor of the interface between DVF and NAIP and successfully alleviated the proinflammatory effect of DVF in colitis. CONCLUSION: Targeting DVF-induced NAIP/NLRC4 inflammasome activation and macrophage pyroptosis ameliorates UC. This finding is of great significance for exploring the gut microbiota-host interactions in UC development and providing new insights for precise treatment.

Aneurysm Infection Caused by Desulfovibrio desulfuricans.

An 84-year-old man in Japan who had undergone endovascular aortic repair 9 years earlier had an infected aneurysm develop. We detected Desulfovibrio desulfuricans MB at the site. The patient recovered after surgical debridement, artificial vessel replacement, and appropriate antimicrobial therapy. Clinicians should suspect Desulfovibrio spp. infection in similar cases.

Desulfovibrio in the Gut: The Enemy within?

Desulfovibrio (DSV) are sulfate-reducing bacteria (SRB) that are ubiquitously present in the environment and as resident commensal bacteria within the human gastrointestinal tract. Though they are minor residents of the healthy gut, DSV are opportunistic pathobionts that may overgrow in the setting of various intestinal and extra-intestinal diseases. An increasing number of studies have demonstrated a positive correlation between DSV overgrowth (bloom) and various human diseases. While the relationship between DSV bloom and disease pathology has not been clearly established, mounting evidence suggests a causal role for these bacteria in disease development. As DSV are the most predominant genera of SRB in the gut, this review summarizes current knowledge regarding the relationship between DSV and a variety of diseases. In this study, we also discuss the mechanisms by which these bacteria may contribute to disease pathology.

Study on the treatment of sulfite wastewater by Desulfovibrio.

In the wet flue gas desulfurization (WFGD) process, SO2 is adsorbed by alkaline liquor to produce alkaline wastewater containing sulfate and sulfite. Although the traditional chemical treatment method can achieve a high removal rate, it consumes a large number of chemicals and yields a large number of low-value by-products. The biological treatment process is a greener and more environmentally friendly treatment method. The current work studies microbial flue gas desulfurization directly using sulfite as the electron acceptor in the reduction process. Desulfovibrio were obtained by isolation and purification, and their growth conditions in sulfite wastewater and desulfurization process conditions were investigated by intermittent and continuous experiments. The results of intermittent experiments indicated that the optimal growth conditions of Desulfovibrio were a temperature of 38 °C, a pH value of 8.0, a COD/SO32- of 2 and that the growth of bacteria would be inhibited at a pH above 9.0 or below 7.3. Furthermore, Desulfovibrio could grow in simulated wastewater with a high SO32- concentration of 8000 mg/L. The results of continuous experiments showed that the removal of sulfite and the recovery of elemental sulfur was realized by a micro-oxygen depletion process, and the removal rate of sulfite of 99%, the yield of elemental sulfur is more than 80% and can reach 90% under the condition of low influent concentration. The bacteria grew well at a temperature of 40 °C and a pH value of the influent water of 7.5. To ensure the treatment effect, the hydraulic retention time (HRT) should be more than doubled for each 1000 mg/L increase in the influent sulfite concentration under the same reflux ratio. When the influent sulfite concentration was 1000 mg/L, 2000 mg/L, 3000 mg/L, and 4000 mg/L, the corresponding HRT was 3.01 h, 6.94 h, 17.4 h, and 31.9 h, respectively. The dominant species in the reactor was Desulfovibrio bacteria at 63.9% abundance. This study demonstrated the feasibility of using sulfite as an electron acceptor for microbial desulfurization, which can optimize the initial process and provide the possibility of treating high-concentration sulfite wastewater.

Anaerobic biodegradation of phenanthrene and pyrene by sulfate-reducing cultures enriched from contaminated freshwater lake sediments.

Our current understanding of the susceptibility of hazardous polycyclic aromatic hydrocarbons (PAHs) to anaerobic microbial degradation is very limited. In the present study, we obtained phenanthrene- and pyrene-degrading strictly anaerobic sulfate-reducing enrichments using contaminated freshwater lake sediments as the source material. The highly enriched phenanthrene-degrading culture, MMKS23, was dominated (98%) by a sulfate-reducing bacterium belonging to the genus Desulfovibrio. While Desulfovibrio sp. was also predominant (79%) in the pyrene-degrading enrichment culture, MMKS44, an anoxygenic purple non-sulfur bacterium, Rhodopseudomonas sp., constituted a significant fraction (18%) of the total microbial community. Phenanthrene or pyrene biodegradation by the enrichment cultures was coupled with sulfate reduction, as evident from near stoichiometric consumption of sulfate and accumulation of sulfide. Also, there was almost complete inhibition of substrate degradation in the presence of an inhibitor of sulfate reduction, i.e., 20 mM MoO42-, in the culture medium. After 180 days of incubation, about 79.40 µM phenanthrene was degraded in the MMKS23 culture, resulting in the consumption of 806.80 µM sulfate and accumulation of 625.80 µM sulfide. Anaerobic pyrene biodegradation by the MMKS44 culture was relatively slow. About 22.30 µM of the substrate was degraded after 180 days resulting in the depletion of 239 µM sulfate and accumulation of 196.90 µM sulfide. Biodegradation of phenanthrene by the enrichment yielded a metabolite, phenanthrene-2-carboxylic acid, suggesting that carboxylation could be a widespread initial step of phenanthrene activation under sulfate-reducing conditions. Overall, this novel study demonstrates the ability of sulfate-reducing bacteria (SRB), dwelling in contaminated freshwater sediments to anaerobically biodegrade three-ringed phenanthrene and highly recalcitrant four-ringed pyrene. Our findings suggest that SRB could play a crucial role in the natural attenuation of PAHs in anoxic freshwater sediments.

A Two-Step Single Plex PCR Method for Evaluating Key Colonic Microbiota Markers in Young Mexicans with Autism Spectrum Disorders: Protocol and Pilot Epidemiological Application.

Many neurological disorders have a distinctive colonic microbiome (CM) signature. Particularly, children with autism spectrum disorders (ASD) exhibit a very dissimilar CM when compared to neurotypical (NT) ones, mostly at the species level. Thus far, knowledge on this matter comes from high-throughput (yet very expensive and time-consuming) analytical platforms, such as massive high-throughput sequencing of bacterial 16S rRNA. Here, pure (260/280 nm, ~1.85) stool DNA samples (200 ng.µL-1) from 48 participants [39 ASD, 9 NT; 3-13 y] were used to amplify four candidate differential CM markers [Bacteroides fragilis (BF), Faecalibacterium prausnitzii (FP), Desulfovibrio vulgaris (DV), Akkermansia muciniphila (AM)], using micro-organism-specific oligonucleotide primers [265 bp (BF), 198 bp (FP), 196 bp (DV), 327 bp (AM)] and a standardized two-step [low (step 1: °Tm-5 °C) to high (stage 2: °Tm-0 °C) astringent annealing] PCR protocol (2S-PCR). The method was sensitive enough to differentiate all CM biomarkers in the studied stool donors [↑ abundance: NT (BF, FP, AM), ASD (DV)], and phylogenetic analysis confirmed the primers' specificity.

A diazotrophy-ammoniotrophy dual growth model for the sulfate reducing bacterium Desulfovibrio vulgaris var. Hildenborough.

Sulfate reducing bacteria (SRB) comprise one of the few prokaryotic groups in which biological nitrogen fixation (BNF) is common. Recent studies have highlighted SRB roles in N cycling, particularly in oligotrophic coastal and benthic environments where they could contribute significantly to N input. Most studies of SRB have focused on sulfur cycling and SRB growth models have primarily aimed at understanding the effects of electron sources, with N usually provided as fixed-N (nitrate, ammonium). Mechanistic links between SRB nitrogen-fixing metabolism and growth are not well understood, particularly in environments where fixed-N fluctuates. Here, we investigate diazotrophic growth of the model sulfate reducer Desulfovibrio vulgaris var. Hildenborough under anaerobic heterotrophic conditions and contrasting N availabilities using a simple cellular model with dual ammoniotrophic and diazotrophic modes. The model was calibrated using batch culture experiments with varying initial ammonium concentrations (0-3000 µM) and acetylene reduction assays of BNF activity. The model confirmed the preferential usage of ammonium over BNF for growth and successfully reproduces experimental data, with notably clear bi-phasic growth curves showing an initial ammoniotrophic phase followed by onset of BNF. Our model enables quantification of the energetic cost of each N acquisition strategy and indicates the existence of a BNF-specific limiting phenomenon, not directly linked to micronutrient (Mo, Fe, Ni) concentration, by-products (hydrogen, hydrogen sulfide), or fundamental model metabolic parameters (death rate, electron acceptor stoichiometry). By providing quantitative predictions of environment and metabolism, this study contributes to a better understanding of anaerobic heterotrophic diazotrophs in environments with fluctuating N conditions.