Dissimilatory Adenosine-5 Research Articles

Molecular dynamics simulations of aqueous systems of inhibitor candidates for adenosine-5’-phosphosufate reductase

Molecular dynamics (MD) simulations were used to evaluate some chelating agents as potential candidates to inhibitors for dissimilatory adenosine-5’-phosphosulfate reductase (APSrAB). Molecular docking methods were used to evaluate the best binding modes of these molecules to the enzyme at two binding sites: of the substrate (enzyme active site) by mean the redocking protocol of substrate; and of one of the [Fe4S4]2+ groups by mean of the clusterization protocol. The best docking poses were selected by criteria such as low energy and RMSD (redocking) and the cluster with the higher number of similar poses (clusterization), which were submitted to MD simulations. RMSD, RDF, and hydrogen bonds results revelated that all ligands left the cube site, while in the active site, some ligands remained in their docking region, pointing to the enzyme active site as the best target for the selected ligands. The binding energy results of ligands hydroxamic acid (HXA) and catechol (CAT) showed that they bonded favorably to the enzyme and key residues of the active site contributed significantly to the protein-ligand bind, indicating HAX and CAT may compete with the substrate for interactions with these residues and displaying potential as candidates for experimental studies about APSrAB inhibitors. Communicated by Ramaswamy H. Sarma

Development of parameters compatible with the CHARMM36 force field for [Fe4S4]2+ clusters and molecular dynamics simulations of adenosine-5’-phosphosulfate reductase in GROMACS 2019

DFT calculations were used to obtain parameters compatible with the CHARMM36 force field for iron-sulfur clusters (Fe-S) of the type [Fe4S4]2+ that are coordinated to dissimilatory adenosine-5’-phosphosulfate reductase (APSrAB). Classical molecular dynamics (MD) simulations were performed on two APSrAB systems to validate the parameters and verify the stability of the studied systems. The time analysis of the parameters inserted into the force field was in reasonable agreement with the experimental X-ray diffraction data. The analysis of the time evolution of the studied systems indicated that these systems and, in particular, the clusters in their respective cavities had a good stability and were in agreement with what was observed in previous works. The parameters obtained provide the basis for the study of APSrAB as well as other systems that contain [Fe4S4]2+ through the CHARMM36 force field.

Microbial communities in semi-consolidated carbonate sediments of the Southwest Indian Ridge

White semi-consolidated carbonate sediments attached to black ferromanganese oxide films were collected approximately 50 km west of a newly discovered hydrothermal field near the Southwest Indian Ridge (SWIR). The biodiversity of the prokaryotic communities within the field was examined using clone library-based culture-independent analysis of the exterior black oxides and the interior white carbonates. Subsequent 16S rRNA gene analysis suggested that Gamma-proteobacteria, Acidobacteria, and Thaumarchaeota members dominated the bacterial and archaeal clone libraries. To further characterize the metabolic processes within the microbial community, analyses of the amoA (coding the alpha subunit of the ammonia monooxygenase for Archaea) and aprA (coding the alpha subunit of the dissimilatory adenosine-5'-phosphosulfate reductase for the sulfate-reducing and sulfur-oxidizing prokaryotes) functional genes were conducted. The functional gene analysis results suggested that Thaumarchaeota and Alphaproteobacteria members were the potential players that participated in N and S cycles in this marine carbonate sedimentary environment. This paper is the first to describe the microbial communities and their potential metabolic pathways within the semi-consolidated carbonate sediments of the SWIR.

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C1 compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.

Desulfatitalea tepidiphila gen. nov., sp. nov., a sulfate-reducing bacterium isolated from tidal flat sediment

A novel sulfate-reducing bacterium, strain S28bF(T), was isolated from tidal flat sediment from Tokyo Bay, Japan. Cells of strain S28bF(T) were rod-shaped (0.5-0.6×1.7-3.8 µm), motile and Gram-stain-negative. For growth, the optimum pH was pH 6.8-7.3 and the optimum temperature was 34-42 °C. Strain S28bF(T) used sulfate and thiosulfate as electron acceptors, but not nitrate. The G+C content of the genomic DNA was 56.6 mol%. The fatty acid profile of strain S28bF(T) was characterized by the presence of anteiso-C(15 : 0) and C(16 : 0) as the major components. Phylogenetic analyses based on genes for 16S rRNA, the alpha subunit of dissimilatory sulfite reductase (dsrA) and adenosine-5'-phosphosulfate reductase (aprA) revealed that the isolated strain belonged to the class Deltaproteobacteria. Its closest relative was Desulfosarcina cetonica DSM 7267(T) with a 16S rRNA gene sequence similarity of 93.3 %. Two other strains, S28OL1 and S28OL2 were also isolated from the same sediment. These strains were closely related to S28bF(T) with 16S rRNA gene sequence similarities of 99 %, and the same physiological characteristics were shared with strain S28bF(T). On the basis of phylogenetic and phenotypic characterization, a novel species in a new genus, Desulfatitalea tepidiphila gen. nov., sp. nov., is proposed to accommodate the strains obtained in this study. The type strain is S28bF(T) ( = NBRC 107166(T) = DSM 23472(T)).

Expression patterns of mRNAs for methanotrophy and thiotrophy in symbionts of the hydrothermal vent mussel Bathymodiolus puteoserpentis.

The hydrothermal vent mussel Bathymodiolus puteoserpentis (Mytilidae) from the Mid-Atlantic Ridge hosts symbiotic sulfur- and methane-oxidizing bacteria in its gills. In this study, we investigated the activity and distribution of these two symbionts in juvenile mussels from the Logatchev hydrothermal vent field (14°45'N Mid-Atlantic Ridge). Expression patterns of two key genes for chemosynthesis were examined: pmoA (encoding subunit A of the particulate methane monooxygenase) as an indicator for methanotrophy, and aprA (encoding the subunit A of the dissimilatory adenosine-5'-phosphosulfate reductase) as an indicator for thiotrophy. Using simultaneous fluorescence in situ hybridization (FISH) of rRNA and mRNA we observed highest mRNA FISH signals toward the ciliated epithelium where seawater enters the gills. The levels of mRNA expression differed between individual specimens collected in a single grab from the same sampling site, whereas no obvious differences in symbiont abundance or distribution were observed. We propose that the symbionts respond to the steep temporal and spatial gradients in methane, reduced sulfur compounds and oxygen by modifying gene transcription, whereas changes in symbiont abundance and distribution take much longer than regulation of mRNA expression and may only occur in response to long-term changes in vent fluid geochemistry.

Effect of the deletion of qmoABC and the promoter-distal gene encoding a hypothetical protein on sulfate reduction in Desulfovibrio vulgaris Hildenborough.

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5'-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the Delta(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments

Multicellular magnetotactic prokaryotes (MMPs) represent highly organized, spherical and motile aggregates of 10-40 bacterial cells containing magnetosomes. Although consisting of different cells, each with its own magnetosomes and flagellation, MMPs orient themselves within a magnetic field and exhibit magnetotaxis. So far, MMPs have only been found in several North and South American coastal lagoons and salt marshes. In the present study, a novel type of MMP was discovered in coastal tidal sand flats of the North Sea. High-resolution scanning electron microscopy revealed the presence of bullet-shaped magnetosomes which were aligned in several parallel chains. Within each aggregate, the magnetosome chains of individual cells were oriented in the same direction. Energy dispersive X-ray (EDX) analysis showed that the magnetosomes are composed of iron sulfide. This particular morphology and arrangement of magnetosomes has previously not been reported for other MMPs. 16S rRNA gene sequence analysis revealed a single phylotype which represented a novel phylogenetic lineage with >or= 4% sequence divergence to all previously described MMP sequences and was related to the dissimilatory sulfate-reducing Desulfosarcina variabilis within the family Desulfobacteraceae of the subphylum Deltaproteobacteria. Fluorescence in situ hybridization with a specific oligonucleotide probe revealed that all MMPs in the tidal flat sediments studied belonged to the novel phylotype. Within each MMP, all bacterial cells showed a hybridization signal, indicating that the aggregates are composed of cells of the same phylotype. Genes for dissimilatory sulfite reductase (dsrAB) and dissimilatory adenosine-5'-phosphate reductase (aprA) could be detected in purified MMP samples, suggesting that MMPs are capable of sulfate reduction. Chemotaxis assays with 41 different test compounds yielded strong responses towards acetate and propionate, whereas other organic acids, alcohols, sugars, sugar alcohols or sulfide did not elicit any response. By means of its coordinated magnetotaxis and chemotaxis, the novel type of MMP is well adapted to the steep chemical gradients which are characteristic for intertidal marine sediments.

Homology Modeling of Dissimilatory APS Reductases (AprBA) of Sulfur-Oxidizing and Sulfate-Reducing Prokaryotes

BackgroundThe dissimilatory adenosine-5′-phosphosulfate (APS) reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers) catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP); in sulfur-oxidizing bacteria (SOB) it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle.Methodology/Principal FindingsFull-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8–99.3% and 89.5–96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 Å with respect to the template structures). Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from.ConclusionsBased on the comparative models, there are no significant structural differences between dissimilatory APS reductases from SRP and SOB; this might be indicative for a similar catalytic process of APS reduction/sulfite oxidation.

Molecular analysis of the diversity of sulfate-reducing and sulfur-oxidizing prokaryotes in the environment, using aprA as functional marker gene.

The dissimilatory adenosine-5'-phosphosulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria.

Molecular analysis of the distribution and phylogeny of dissimilatory adenosine-5′-phosphosulfate reductase-encoding genes (aprBA) among sulfur-oxidizing prokaryotes

Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Phylogeny of the alpha and beta subunits of the dissimilatory adenosine-5′-phosphosulfate (APS) reductase from sulfate-reducing prokaryotes – origin and evolution of the dissimilatory sulfate-reduction pathway

Newly developed PCR assays were used to PCR-amplify and sequence fragments of the dissimilatory adenosine-5'-phosphosulfate (APS) reductase genes (aprBA) comprising nearly the entire gene locus (2.2-2.4 kb, equal to 92-94 % of the protein coding sequence) from 75 sulfate-reducing prokaryotes (SRP) of a taxonomically wide range. Comparative phylogenetic analysis included all determined and publicly available AprBA sequences from SRP and selected homologous sequences of sulfur-oxidizing bacteria (SOB). The almost identical AprB and AprA tree topologies indicated a shared evolutionary path for the aprBA among the investigated SRP by vertical inheritance and concomitant lateral gene transfer (LGT). The topological comparison of AprB/A- and 16S rRNA gene-based phylogenetic trees revealed novel LGT events across the SRP divisions. Compositional gene analysis confirmed Thermacetogenium phaeum to be the first validated strain affected by a recent lateral transfer of aprBA as a putative effect of long-term co-cultivation with a Thermodesulfovibrio species. Interestingly, the Apr proteins of SRP and SOB diverged into two phylogenetic lineages, with the SRP affiliated with the green sulfur bacteria, e.g. Chlorobaculum tepidum, while the Allochromatium vinosum-related sequences formed a distinct group. Analysis of genome data indicated that this phylogenetic separation is also reflected in the differing presence of the putative proteins functionally associated with Apr, QmoABC complex (quinone-interacting membrane-bound oxidoreductase) and AprM (transmembrane protein). Scenarios for the origin and evolution of the dissimilatory APS reductase are discussed within the context of the dissimilatory sulfite reductase (DsrAB) phylogeny, the appearance of QmoABC and AprM in the SRP and SOB genomes, and the geochemical setting of Archean Earth.

Quantification of Sulfate-reducing Bacteria in Industrial Wastewater, by Real-time Polymerase Chain Reaction (PCR) Using dsrA and apsA Genes

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5'-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 x 10(2) and 9.5 x 10(6) copies/microL and between 1.2 x 10(3) and 1.2 x 10(7) copies/microL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10(7)-10(8) targets mL(-1)) and lower during the winter (about 10(4)-10(5) targets mL(-1)). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.