Dissimilatory Sulfate Reduction Pathway Research Articles

Anaerobic ammonium oxidation coupled with sulfate reduction links nitrogen with sulfur cycle

Sulfate-dependent ammonium oxidation (Sulfammox) is a critical process linking nitrogen and sulfur cycles. However, the metabolic pathway of microbes driven Sulfammox is still in suspense. The study demonstrate that ammonium was not consumed with sulfate as the sole electron acceptor during long-term enrichment, probably due to inhibition from sulfide accumulation, while ammonium was removed at ∼ 10 mg N/L/d with sulfate and nitrate as electron acceptors. Ammonium and sulfate were converted into nitrogen gas, sulfide, and elemental sulfur. Sulfammox was mainly performed by Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida, both of which encoded ammonium oxidation pathway and dissimilatory sulfate reduction pathway. Not sulfide-driven autotrophic denitrifiers but Candidatus Kuenenia stuttgartiensis converted nitrate to nitrite with sulfide. The results of this study reveal the specialized metabolism of Sulfammox bacteria (Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida) and provide insight into microbial relationships during the nitrogen and sulfur cycles.

Characteristics and mechanisms of phosphine production in sulfur-based constructed wetlands

Phosphine (PH3) is an important contributor to the phosphorus cycle and is widespread in various environments. However, there are few studies on PH3 in constructed wetlands (CWs). In this study, lab-scale CWs and batch experiments were conducted to explore the characteristics and mechanisms of PH3 production in sulfur-based CWs. The results showed that the PH3 release flux of sulfur-based CWs varied from 0.86±0.04 ng·m−2·h−1 to 1.88±0.09 ng·m−2·h−1. The dissolved PH3 was the main PH3 form in CWs and varied from 2.73 μg·L−1 to 4.08 μg·L−1. The matrix-bound PH3 was a staging reservoir for PH3 and increased with substrate depth. In addition, the sulfur-based substrates had a significant improvement on PH3 production. Elemental sulfur is more conducive to PH3 production than pyrite. Moreover, there was a significant positive correlation between PH3 production, the dsrB gene, and nicotinamide adenine dinucleotide (NADH). NADH might catalyze the phosphate reduction process. And the final stage of the dissimilatory sulfate reduction pathway driven by the dsrB gene might also provide energy for phosphate reduction. The migration and transformation of PH3 increased the available P (Resin-P and NaHCO3-P) from 35 % to 56 % in sulfur-based CW, and the P adsorption capacity was improved by 12 %. The higher proportion of available P increased the plant uptake rate of P by 17 %. This study improves the understanding of the phosphorus cycle in sulfur-based CW and provides new insight into the long-term stable operation of CWs.

A bioaugmentation strategy to recover methane production under sulfate-stressed conditions: Highlights on targeted sulfate-reducing bacteria and DIET-related species

Bioaugmentation has been recognized as a key strategy to improve the anaerobic digestion efficiency in organic waste treatment. Methanosarcina barkeri possesses direct interspecies electron transfer capability, a characteristic that allows it to outcompete other unwanted species such as sulfate-reducing bacteria. This study investigated the effects of bioaugmentation with Methanosarcina barkeri DSM800 on two continuous-stirred tank reactors fed with a sulfate-rich feedstock. One of the two reactors was supplemented with magnetite to facilitate direct interspecies electron transfer. Time series quantitative polymerase chain reactions were performed to evaluate the absolute abundance of crucial species, including the augmented Methanosarcina. Results showed increased and stabilized methane production of 22% and 21% in the reactor amended with magnetite and in the control reactor, respectively. Moreover, volatile fatty acids were almost completely consumed in the magnetite-supplemented reactor. The quantitative polymerase chain reaction was used to analyze the abundance of targeted species in response to bioaugmentation. Specifically, Methanosarcina barkeri was not retained in either reactor after one hydraulic retention time. Direct interspecies electron transfer-associated microorganisms showed opposite trends in the two reactors, highlighting the different interactions with Methanosarcina barkeri in the presence and absence of magnetite. Sulfate-reducing bacteria following the dissimilatory sulfate reduction pathway exhibited an opposite behavior in the reactor amended with magnetite, in contrast to those employing the assimilatory sulfate reduction pathway. Overall, the study demonstrated that bioaugmentation with exogenous archaea can considerably alter the microbial community, but the introduced species is not able to establish itself in a stable microbiome. In addition, the strategy could be further tested to control H2S production in real-world waste treatment scenarios. Quantitative polymerase chain reaction proved to be a useful tool for monitoring changes in the absolute abundance of microorganisms in bioreactors, implementing effective monitoring and control strategies to improve overall system performance.

The Isolation of Anaerobic and Facultative Anaerobic Sulfate-Reducing Bacteria (SRB) and a Comparison of Related Enzymes in Their Sulfate Reduction Pathways.

Sulfate-reducing bacteria (SRB) are an important group of microorganisms that cause microbial corrosion. In this study, culturable SRB were isolated and identified from the inner rust layer of three kinds of steel and from sediments, and a comparison of amino acid sequences encoding related enzymes in the sulfate reduction pathway between anaerobic and facultative anaerobic SRB strains was carried out. The main results are as follows. (1) Seventy-seven strains were isolated, belonging to five genera and seven species, with the majority being Desulfovibrio marinisediminis. For the first time, Holodesulfovibrio spirochaetisodalis and Acinetobacter bereziniae were separated from the inner rust layer of metal, and sulfate reduction by A. bereziniae, Virgibacillus dokdonensis, and Virgibacillus chiguensis, etc., was also demonstrated for the first time. (2) Three strains of strictly anaerobic bacteria and four strains of facultative anaerobic bacteria were screened from seven bacterial strains. (3) Most of the anaerobic SRB only contained enzymes for the dissimilatory sulfate reduction pathway, while those of facultative anaerobic bacteria capable of producing hydrogen sulfide included two possible ways: containing the related enzymes from the dissimilatory pathway only, or containing enzymes from both dissimilatory and assimilation pathways. This study newly discovered that some bacterial genera exhibit sulfate reduction ability and found that there are differences in the distribution of enzymes related to the sulfate reduction pathway between anaerobic and facultative anaerobic SRB type trains, providing a basis for the development and utilization of sulfate-reducing bacterial resources and furthering our understanding of the metabolic mechanisms of SRB.

Microbial electrolysis cell as an effective tool for mitigating the toxic effects of Cu2+ on microorganisms in the acid mine drainage treatment: Efficacy and mechanisms

It is poorly understood how the mechanism of heavy metals affect sulfate-reducing bacteria (SRB) in microbial electrolysis cell (MEC) system, however, this is essential to improve the effectiveness of sulfate reduction in acid mine drainage (AMD). This study evaluated the impact of Cu2+ on MEC performance using SRBs enriched biocathode. The sulfate reduction rate and pathway, the fate of Cu, as well as the succession of microbial community were investigated. Results showed that MEC performance was optimized at influent Cu2+ concentration of 20 mg/L, while slight inhibition was observed at 40 mg/L. The removal of Cu2+ was accomplished through bioprecipitation and biosorption, forming CuS and Cu(OH)2. However, MEC performance was inhibited at higher Cu2+ concentrations (60 mg/L) due to damaged cell structure, increased charge transfer internal resistance, and altered microbial communities. Specifically, the relative abundance of Firmicutes and Bacteroidota decreased, and functional genes related to sulfate reduction metabolism were reduced, leading to impairment of the assimilatory and dissimilatory sulfate reduction pathways. This knowledge provides revealing insights and theoretical support for developing a feasible approach to realize the treatment of wastewater containing multiple pollutants.

The link between increased Desulfovibrio and disease severity in Parkinson’s disease

Parkinson's disease (PD), a progressive and incurable neurodegenerative disease, has taken a huge economic toll and medical burden on our society. Increasing evidence has shown a strong link between PD and the gut microbiome, but studies on the relationship between the gut microbiome and the severity of PD are limited. In this study, 90 fecal samples were collected from newly diagnosed and untreated patients with PD (n = 47) and matched healthy control subjects (n = 43). The 16S rRNA amplicon and shotgun metagenomic sequencing was performed, aiming to uncover the connection between the gut microbiome and disease severity in PD. The results showed that Desulfovibrio was significantly increased in PD compared to healthy controls and positively correlated with disease severity. The increase in Desulfovibrio was mainly driven by enhanced homogeneous selection and weakened drift. Moreover, through metagenome-assembled genomes (MAGs) analysis, a Desulfovibrio MAG (MAG58) was obtained which was also positively correlated with disease severity. MAG58 possesses a complete assimilatory sulfate reduction pathway and a near-complete dissimilatory sulfate reduction pathway to produce hydrogen sulfide which may influence the development of PD. Based on these results, a potential pathogenic mechanism was presented to illustrate how the increased Desulfovibrio accelerates the development of PD by producing excessive hydrogen sulfide. The present study highlighted the vital role of Desulfovibrio in the development of PD, which may provide a new target for the diagnosis and treatment of PD. KEY POINTS: • The evidence for the link between increased Desulfovibrio and disease severity in PD • A Desulfovibrio MAG was obtained which was correlated with PD • A model was presented to illustrate how increased Desulfovibrio causes PD.

What Controls the Sulfur Isotope Fractionation during Dissimilatory Sulfate Reduction?

Sulfate often behaves conservatively in the oxygenated environments but serves as an electron acceptor for microbial respiration in a wide range of natural and engineered systems where oxygen is depleted. As a ubiquitous anaerobic dissimilatory pathway, therefore, microbial reduction of sulfate to sulfide has been of continuing interest in the field of microbiology, ecology, biochemistry, and geochemistry. Stable isotopes of sulfur are an effective tool for tracking this catabolic process as microorganisms discriminate strongly against heavy isotopes when cleaving the sulfur-oxygen bond. Along with its high preservation potential in environmental archives, a wide variation in the sulfur isotope effects can provide insights into the physiology of sulfate reducing microorganisms across temporal and spatial barriers. A vast array of parameters, including phylogeny, temperature, respiration rate, and availability of sulfate, electron donor, and other essential nutrients, has been explored as a possible determinant of the magnitude of isotope fractionation, and there is now a broad consensus that the relative availability of sulfate and electron donors primarily controls the magnitude of fractionation. As the ratio shifts toward sulfate, the sulfur isotope fractionation increases. The results of conceptual models, centered on the reversibility of each enzymatic step in the dissimilatory sulfate reduction pathway, are in qualitative agreement with the observations, although the underlying intracellular mechanisms that translate the external stimuli into the isotopic phenotype remain largely unexplored experimentally. This minireview offers a snapshot of our current understanding of the sulfur isotope effects during dissimilatory sulfate reduction as well as their potential quantitative applications. It emphasizes the importance of sulfate respiration as a model system for the isotopic investigation of other respiratory pathways that utilize oxyanions as terminal electron acceptors.

Stability improvement and the mechanism of a microbial electrolysis cell biocathode for treating wastewater containing sulfate by quorum sensing

The treatment of sulfate-containing wastewater by microbial electrolysis cells (MEC) is a feasible method, but the biocathode suffers from poor microbial stability. The phenomenon of quorum sensing (QS) has offered the opportunity to overcome these weaknesses. Nevertheless, it has not been well evaluated how QS improves microbial stability performance and which enzymes and genes influence microbial activity. In this study, N-butyryl-l-Homoserine lactone (C4-HSL) was added as a typical QS signaling molecule to the biocathode in MEC. Sulfate reduction efficiency, microbial community structure, and related functional genes were analyzed comprehensively. The results showed that the sulfate reduction efficiency and biocathode stability were higher under the regulation of C4-HSL. Results indicated that the number of microorganisms on the electrode surface was more than that of the control group, and the percentage of live cells was 22% higher after the regulation by C4-HSL. In addition, the C4-HSL regulation stimulated the growth of the hydrogen producer (Desulfomicrobium), Homoacetogens (Acetoanaerobium), and part of Anaerobic fermentation bacteria (Macellibacteroides and Lentimicrobium). Metagenomic sequencing elucidated the phenomenon of QS mainly through an exponential increase in autoinducer-producer-related genes and cysteine/methionine metabolism pathways. The regulation by C4-HSL mainly affected genes controlling surface proteins and biofilm formation, which resulted in higher activity and more stability of biofilms. And the relative abundance of functional genes associated with sulfate reduction metabolism was increased, which positively affected the assimilatory sulfate reduction and dissimilatory sulfate reduction pathways. These findings reveal the potential and mechanisms of the QS signaling molecule in enhancing MEC for sulfate-containing wastewater treatment.

Early-stage response in anaerobic bioreactors due to high sulfate loads: Hydrogen sulfide yield and other organic volatile sulfur compounds as a sign of microbial community modifications

In this work, the early-stage response of six lab-scale biogas bioreactors fed with different amounts of a sulfate-rich organic agro-industrial effluent was investigated. Biogas characterization, gas chromatography selective for sulfur compounds and high-throughput sequencing of 16S rRNA gene were performed. Hydrogen sulfide (H2S) yield went from transient to steady state in ∼ 2 weeks for all the studied conditions. In addition, volatile sulfur compounds (VSCs), like methanethiol (MeSH) and dimethyl sulfide (DMS), were generated at high sulfate loads. Changes were evidenced in the microbial community structures, with a higher abundance of genes involved in the dissimilatory sulfate-reduction pathway in high loaded sulfate bioreactors, as determined by PICRUSt analysis. Principal component analysis (PCA) and correlation analyses evidenced strong relationships between H2S, VSCs and the microbial community. Sulfate-reducing bacteria (SRB) like Desulfocarbo, Desulfocella and Desulfobacteraceae might be possibly linked with methylation processes of H2S.

Genetic Potential of Dissulfurimicrobium hydrothermale, an Obligate Sulfur-Disproportionating Thermophilic Microorganism

The biochemical pathways of anaerobic sulfur disproportionation are only partially deciphered, and the mechanisms involved in the first step of S0-disproportionation remain unknown. Here, we present the results of sequencing and analysis of the complete genome of Dissulfurimicrobium hydrothermale strain Sh68T, one of two strains isolated to date known to grow exclusively by anaerobic disproportionation of inorganic sulfur compounds. Dissulfurimicrobium hydrothermale Sh68T is a motile, thermophilic, anaerobic, chemolithoautotrophic microorganism isolated from a hydrothermal pond at Uzon caldera, Kamchatka, Russia. It is able to produce energy and grow by disproportionation of elemental sulfur, sulfite and thiosulfate. Its genome consists of a circular chromosome of 2,025,450 base pairs, has a G + C content of 49.66% and a completion of 97.6%. Genomic data suggest that CO2 assimilation is carried out by the Wood–Ljungdhal pathway and that central anabolism involves the gluconeogenesis pathway. The genome of strain Sh68T encodes the complete gene set of the dissimilatory sulfate reduction pathway, some of which are likely to be involved in sulfur disproportionation. A short sequence protein of unknown function present in the genome of strain Sh68T is conserved in the genomes of a large panel of other S0-disproportionating bacteria and was absent from the genomes of microorganisms incapable of elemental sulfur disproportionation. We propose that this protein may be involved in the first step of elemental sulfur disproportionation, as S0 is poorly soluble and unable to cross the cytoplasmic membrane in this form.

An assimilatory sulfite reductase, CysI, negatively regulates the dormancy of Microbulbifer aggregans CCB-MM1T.

Sulfur is one of the common and essential elements of all life. Sulfate, which is a major source of sulfur, plays an important role in synthesizing sulfur-containing amino acids, such as cysteine and methionine, organic compounds essential to all living organisms. Some investigations reported that the assimilatory sulfate reduction pathway (ASRP) involved in cysteine synthesis is crucial to entering bacterial dormancy in pathogens. Our previous investigation reported that the halophilic marine bacterium, Microbulbifer aggregans CCB-MM1T , possesses an ASRP and the dissimilatory sulfate reduction pathway (DSRP). The bacterium might use DSRP to generate energy required for entering its dormant. However, the role of the ASRP in the dormancy of M. aggregans CCB-MM1T was so far unknown. In this study, we found that genes involved in ASRP were downregulated in the dormancy. The disruption of the gene encoding an assimilatory sulfite reductase, cysI, suppressed a completely dormant state under low nutrient conditions. In addition, the cysI mutant showed cell aggregation at the middle-exponential phase under high nutrient conditions, indicating that the mutation might be stimulated to enter the dormancy. The wild-type phenotype of the bacterium was recovered by the addition of cysteine. These results suggested that cysteine concentration may play an important role in inducing the dormancy of M. aggregans.

Substrate competition and microbial function in sulfate-reducing internal circulation anaerobic reactor in the presence of nitrate

Nitrate and sulfate often coexist in organic wastewater. In this study, an internal circulation anaerobic reactor was conducted to investigate the impact of nitrate on sulfate reduction. The results showed that sulfate reduction rate dropped from 78.4% to 41.4% at NO3- /SO42- ratios ranging from 0 to 1.03, largely attributed to the inactivity of acetate-utilizing sulfate-reducing bacteria (SRB) and preferential usage of nitrate of propionate-utilizing SRB. Meanwhile, high nitrate removal efficiency was maintained and COD removal efficiency increased with nitrate addition. Enhancement of propionate and butyrate degradation based on Modified Gompertz model and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis. Moreover, nitrate triggered the shift of microbial community and function. Twelve genera affiliated to Firmicutes, Bacteroidetes and Proteobacteria were identified as keystone genera via network analysis, which kept functional stability of the bacterial community responding to nitrate stress. Increased nitrate inhibited Desulfovibrio, but promoted the growth of Desulforhabdus. Both the predicted functional genes associated with assimilatory sulfate reduction pathway (cysC and cysNC) and dissimilatory sulfate reduction pathway (aprA, aprB, dsrA and dsrB) exhibited negative relationship with nitrate addition.

Soil Metagenomics Reveals Effects of Continuous Sugarcane Cropping on the Structure and Functional Pathway of Rhizospheric Microbial Community.

The continuous cropping of plants can result in the disruption of the soil microbial community and caused significant declines in yields. However, there are few reports on the effects of continuous cropping of sugarcane on the microbial community structure and functional pathway. In the current study, we analyzed the structural and functional changes of microbial community structure in the rhizospheric soil of sugarcane in different continuous cropping years using Illumina Miseq high-throughput sequencing and metagenomics analysis. We collected rhizosphere soils from fields of no continuous cropping history (NCC), 10 years of continuous cropping (CC10), and 30 years of continuous cropping (CC30) periods in the Fujian province. The results demonstrated that continuous sugarcane cropping resulted in significant changes in the physicochemical properties of soil and the composition of soil bacterial and fungal communities. With the continuous cropping, the crop yield dramatically declined from NCC to CC30. Besides, the redundancy analysis (RDA) of the dominant bacterial and fungal phyla and soil physicochemical properties revealed that the structures of the bacterial and fungal communities were mainly driven by pH and TS. Analysis of potential functional pathways during the continuous cropping suggests that different KEGG pathways were enriched in different continuous cropping periods. The significant reduction of bacteria associated with rhizospheric soil nitrogen and sulfur cycling functions and enrichment of pathogenic bacteria may be responsible for the reduction of effective nitrogen and total sulfur content in rhizospheric soil of continuous sugarcane as well as the reduction of sugarcane yield and sugar content. Additionally, genes related to nitrogen and sulfur cycling were identified in our study, and the decreased abundance of nitrogen translocation genes and AprAB and DsrAB in the dissimilatory sulfate reduction pathway could be the cause of declined biomass. The findings of this study may provide a theoretical basis for uncovering the mechanism of obstacles in continuous sugarcane cropping and provide better guidance for sustainable development of the sugarcane.

Metagenomic insights into aniline effects on microbial community and biological sulfate reduction pathways during anaerobic treatment of high-sulfate wastewater

For comprehensive insights into the change of sulfate reduction pathway responding to the toxic stress and the shift of microbial community and performance of sulfate reduction, we built a laboratory-scale expanded granular sludge bed reactor (EGSB) treating high-sulfate wastewater with elevated aniline concentrations from 0 to 480 mg/L. High-throughput sequencing and metagenomic approaches were applied to decipher the molecular mechanisms of sulfate reduction under aniline stress through taxonomic and functional profiles. The increasing aniline in the anaerobic system induced the accumulation of volatile fatty acids (VFA), further turned the bioreactor into acidification, which was the principal reason for the deterioration of system performance and finally resulted in the accumulation of toxic free sulfide. Moreover, aniline triggered the change of bacterial community and genes relating to sulfate reduction pathways. The increase of aniline from 0 to 320 mg/L enriched total sulfate-reducing bacteria (SRB), and the most abundant genus was Desulfomicrobium, accounting for 66.85–91.25% of total SRB. The assimilatory sulfate reduction pathway was obviously inhibited when aniline was over 160 mg/L, while genes associated with dissimilatory sulfate reduction pathways all exhibited an upward tendency with the increasing aniline content. The enrichment of aniline-resistant SRB (e.g. Desulfomicrobium) carrying genes associated with the dissimilatory sulfate reduction pathway also confirmed the underlying mechanism that sulfate reduction turned into dissimilation under high aniline condition. Taken together, these results comprehensively provided solid evidence for the effects of aniline on the biological sulfate reduction processes treating high-sulfate wastewater and the underlying molecular mechanisms which may highlight the important roles of SRB and related sulfate reduction genes during treatment.

Metagenomic analysis suggests broad metabolic potential in extracellular symbionts of the bivalve Thyasira cf. gouldi

BackgroundNext-generation sequencing has opened new avenues for studying metabolic capabilities of bacteria that cannot be cultured. Here, we provide a metagenomic description of chemoautotrophic gammaproteobacterial symbionts associated with Thyasira cf. gouldi, a sediment-dwelling bivalve from the family Thyasiridae. Thyasirid symbionts differ from those of other bivalves by being extracellular, and recent work suggests that they are capable of living freely in the environment.ResultsThyasira cf. gouldi symbionts appear to form mixed, non-clonal populations in the host, show no signs of genomic reduction and contain many genes that would only be useful outside the host, including flagellar and chemotaxis genes. The thyasirid symbionts may be capable of sulfur oxidation via both the sulfur oxidation and reverse dissimilatory sulfate reduction pathways, as observed in other bivalve symbionts. In addition, genes for hydrogen oxidation and dissimilatory nitrate reduction were found, suggesting varied metabolic capabilities under a range of redox conditions. The genes of the tricarboxylic acid cycle are also present, along with membrane bound sugar importer channels, suggesting that the bacteria may be mixotrophic.ConclusionsIn this study, we have generated the first thyasirid symbiont genomic resources. In Thyasira cf. gouldi, symbiont populations appear non-clonal and encode genes for a plethora of metabolic capabilities; future work should examine whether symbiont heterogeneity and metabolic breadth, which have been shown in some intracellular chemosymbionts, are signatures of extracellular chemosymbionts in bivalves.

Expanding anaerobic alkane metabolism in the domain of Archaea.

Methanogenesis and anaerobic methane oxidation through methyl-coenzyme M reductase (MCR) as a key enzyme have been suggested to be basal pathways of archaea1. How widespread MCR-based alkane metabolism is among archaea, where it occurs and how it evolved remain elusive. Here, we performed a global survey of MCR-encoding genomes based on metagenomic data from various environments. Eleven high-quality mcr-containing metagenomic-assembled genomes were obtained belonging to the Archaeoglobi in the Euryarchaeota, Hadesarchaeota and different TACK superphylum archaea, including the Nezhaarchaeota, Korarchaeota and Verstraetearchaeota. Archaeoglobi WYZ-LMO1 and WYZ-LMO3 and Korarchaeota WYZ-LMO9 encode both the (reverse) methanogenesis and the dissimilatory sulfate reduction pathway, suggesting that they have the genomic potential to couple both pathways in individual organisms. The Hadesarchaeota WYZ-LMO4-6 and Archaeoglobi JdFR-42 encode highly divergent MCRs, enzymes that may enable them to thrive on non-methane alkanes. The occurrence of mcr genes in different archaeal phyla indicates that MCR-based alkane metabolism is common in the domain of Archaea.

Deconstructing the Dissimilatory Sulfate Reduction Pathway: Isotope Fractionation of a Mutant Unable of Growth on Sulfate.

The sulfur isotope record provides key insight into the history of Earth's redox conditions. A detailed understanding of the metabolisms driving this cycle, and specifically microbial sulfate reduction (MSR), is crucial for accurate paleoenvironmental reconstructions. This includes a precise knowledge of the step-specific sulfur isotope effects during MSR. In this study, we aim at resolving the cellular-level fractionation factor during dissimilatory sulfite reduction to sulfide within MSR, and use this measured isotope effect as a calibration to enhance our understanding of the biochemistry of sulfite reduction. For this, we merge measured isotope effects associated with dissimilatory sulfite reduction with a quantitative model that explicitly links net fractionation, reaction reversibility, and intracellular metabolite levels. The highly targeted experimental aspect of this study was possible by virtue of the availability of a deletion mutant strain of the model sulfate reducer Desulfovibrio vulgaris (strain Hildenborough), in which the sulfite reduction step is isolated from the rest of the metabolic pathway owing to the absence of its QmoABC complex (ΔQmo). This deletion disrupts electron flux and prevents the reduction of adenosine phosphosulfate (APS) to sulfite. When grown in open-system steady-state conditions at 10% maximum growth rate in the presence of sulfite and lactate as electron donor, sulfur isotope fractionation factors averaged −15.9‰ (1 σ = 0.4), which appeared to be statistically indistinguishable from a pure enzyme study with dissimilatory sulfite reductase. We coupled these measurements with an understanding of step-specific equilibrium and kinetic isotope effects, and furthered our mechanistic understanding of the biochemistry of sulfite uptake and ensuing reduction. Our metabolically informed isotope model identifies flavodoxin as the most likely electron carrier performing the transfer of electrons to dissimilatory sulfite reductase. This is in line with previous work on metabolic strategies adopted by sulfate reducers under different energy regimes, and has implications for our understanding of the plasticity of this metabolic pathway at the center of our interpretation of modern and palaeo-environmental records.

Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia.

SummaryThe sulfate‐dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon‐rich environments under different temperature regimes: ANME‐1a/HotSeep‐1 (60°C), ANME‐1a/Seep‐SRB2 (37°C) and ANME‐2c/Seep‐SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME‐2c encodes all enzymes, while ANME‐1a lacks the gene for N5,N10‐methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c‐type cytochromes, some predicted to be extracellular. ANME‐2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME‐1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire‐like structures and heme‐rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate‐dependent AOM, and that both partners synthesize molecules to enable it.

Quantification and isotopic analysis of intracellular sulfur metabolites in the dissimilatory sulfate reduction pathway

Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in 34S and producing sulfide that is depleted in 34S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfur isotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellular mechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopic ratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis. Intracellular sulfate and APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulates in the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in 34S. The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfate and sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the net fractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This ‘reservoir effect’ diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations. Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the overall (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for the well-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while at the same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in this relationship. Our approach, together with ongoing investigations of the kinetic isotope fractionation by key enzymes in the sulfate reduction pathway, should provide an empirical basis for a quantitative model relating the magnitude of microbial isotope fractionation to their environmental and physiological controls.

Integrated metagenomic and metaproteomic analyses of an ANME‐1‐dominated community in marine cold seep sediments

Sulfate-reducing methanotrophy by anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) is a major biological sink of methane in anoxic methane-enriched marine sediments. The physiology of a microbial community dominated by free-living ANME-1 at 14-16 cm below the seafloor in the G11 pockmark at Nyegga was investigated by integrated metagenomic and metaproteomic approaches. Total DNA was subjected to 454-pyrosequencing (829 527 reads), and 16.6 Mbp of sequence information was assembled into 27352 contigs. Taxonomic analysis supported a high abundance of Euryarchaea (70%) with 66% of the assembled metagenome belonging to ANME-1. Extracted sediment proteins were separated in two dimensions and subjected to mass spectrometry (LTQ-Orbitrap XL). Of 356 identified proteins, 245 were expressed by ANME-1. These included proteins for cold-adaptation and production of gas vesicles, reflecting both the adaptation of the ANME-1 community to a permanently cold environment and its potential for positioning in specific sediment depths respectively. In addition, key metabolic enzymes including the enzymes in the reverse methanogenesis pathway (except N(5) ,N(10) -methylene-tetrahydromethanopterin reductase), heterodisulfide reductases and the F(420) H(2) :quinone oxidoreductase (Fqo) complex were identified. A complete dissimilatory sulfate reduction pathway was expressed by sulfate-reducing Deltaproteobacteria. Interestingly, an APS-reductase comprising Gram-positive SRB and related sequences were identified in the proteome. Overall, the results demonstrated that our approach was effective in assessing in situ metabolic processes in cold seep sediments.