Investigating Pathways of Sulfidogenesis in Thiosulfate-Reducing Halanaerobium Species Annette De Capite Department of Microbiology, University of Oklahoma Sulfidogenic Bacteria Sulfate-reducing bacteria 2- • Sulfate ( SO4 ) 2- • Sulfite ( SO3 ) Sulfur -reducing bacteria Sulfide • Elemental sulfur ( S8) HS- / H S Thiosulfate-reducing bacteria (aq) 2 (g) 2- Thiosulfate ( S2O3 ) Observations -4 M Cl -, 49oC Halanaerobium! SRB? Anaerobic fermenters common to high salt, high temperature oil production waters. Middle East Field 16S Results (OU Biocorrosion Center) Materials Sample [Cl -] (Molar) Temperature Angola production water 1.5 49 oC Middle East Field production water 2.5 49 oC European Oil Reservoir 2.5 37 oC High Temperature, High Salt Production Waters Enrichments established under fermentative and thiosulfate -reducing conditions. Halanaerobiaceae Isolation Results 23 sulfidogenic isolates obtained! Halanaerobium Genus Genus Genus Novel Genus? TR denotes species confirmed for thiosulfate reduction Bold strains represent species used in this study Genus Halanaerobium Firmicutes Obligate Anaerobes Obligate halophiles Isolated from saline production waters, brine lakes, and microbial mats 1 µm Fermentative metabolism Do not reduce sulfate! species produce sulfide from the reduction of thiosulfate and sulfur. Halanaerobium kushneri Sources of Sulfate and Thiosulfate Sulfate - 2- Production waters HS S2O3 SO4 Seawater Interconversion to other sulfur anions Thiosulfate 2- 2- SO4 S2O3 Production waters (Mineral) Seawater 2- 2- Oxidation of SO4 S2O3 sulfides (Seawater) HS Molecular Detection of Sulfidogenic Bacteria 16S rRNA gene Functional genes Applications Caveats Assess if microbial Does not confirm viability of community has the bacteria or activity of genetic potential for sulfidogenic enzymes. sulfidogenesis. Cultivation and physiology! Gene Targets for SRB Amplification ≠ Enzymatic Activity aprBA dsrABD 2- * 2- - SO4 APS SO3 HS Sulfate Sulfite Sulfide APS Dissimilatory Reductase Sulfite Reductase Gene Targets for TRB Thiosulfate * Thiosulfate Rhodanese Reductase disproportionation 2- 2- 2- S2O3 S2O3 S2O3 aprBA rdl phs Molybdopterin sat protein 2- 2- - - SCN SO3 SO3 HS HS SO Sulfite Sulfite Sulfide Thiocyanate Sulfide Sulfate Sulfite Reductases Proposed to be involved in thiosulfate -reduction of HS- Halanaerobium species. dsrABD or asrABC Sulfide Investigating Enzymatic Pathways 1. Assess if other sulfur anions are reduced 2. Evaluate rhodanese activity 3. Identify end products of thiosulfate reduction 4. Determine sulfide/thiosulfate ratio 5. Genomic analysis SulfidogenesisZB2A: Sulfidogenesis in H. vreelandiifrom Sulfur Anions * Sulfide produced from reduction of S O 2- and S * 2 3 8 Thiosulfate reduction also confirmed in H. congolense and OKU7 *Significantly different compared to fermentative, Student’s t -test, p-value < 0.05. Growth of H. vreelandii in the Presence of Sulfur Anions Similar results observed for H.congolense and OKU7 The Effect of Sulfite on Growth of H. vreelandii Growth is improved as sulfite concentration decreases Investigating Enzymatic Pathways 1. Assess if other sulfur anions are reduced • Elemental sulfur and thiosulfate are reduced to sulfide. • Sulfite inhibits growth 2. Evaluate rhodanese activity Rhodanese Activity: Quantification of Thiocyanate Iron-thiocyanate complex Measure absorbance of Thiocyanate supernatant at 470 nm Ferric nitrate reagent Thiocyanate can only be produced from rhodanese! Rhodanese activity observed in all Halanaerobium strains tested! Rhodanese Activity in H. vreelandii 2- 2- S2O3 SO3 (Inhibitory) CN SCN * Thiocyanate is only detected when culture is amended with cyanide. Investigating Enzymatic Pathways 2. Evaluate rhodanese activity • Both sulfidogenic and non-sulfidogenic species of Halanaerobium possess rhodanese activity. • Only a fraction of the predicted sulfite is detected in the rhodanese assay. ° Where does the sulfite go? 3. Identify end products of thiosulfate reduction 2- Fate of Sulfur Anions S2O3 CN Sulfate was not Thiosulfate SCN TThiosulfatehiosulfate produced. Thiocyanate Rhodanese disproportionationdisproportionation Sulfide was generated rdl 2- SO4 as an end product of Thiosulfate reductase Sulfate thiosulfate reduction phs Thiocyanate was TThiosulfatehiosulfate disproportionation disproportionation 2- produced when cyanide SO3 or Sulfite was present. (Inhibitory) Thiosulfate reductase phs Sulfite was not detected Sulfite Reductases in vivo, but detected in dsr or ‘ asr ’ low quantities in the HS- rhodanese assay. Sulfide Investigating Enzymatic Pathways 3. Identify end products of thiosulfate reduction • Thiosulfate disproportionation can be ruled out as an operable pathway. • Rhodanese and thiosulfate reductase cannot not be excluded. • Sulfite must be metabolized to prevent growth inhibition 4. Determine sulfide/thiosulfate ratio 2- Thiosulfate: Sulfide Ratio 1 S2O3 Thiosulfate SCN For every molecule of Rhodanese Thiocyanate thiosulfate reduced, two rdl molecules of sulfide are Thiosulfate reductase phs generated (2:1). Thiosulfate reductase Suggests that both phs 2- thiosulfate and sulfite SO3 reductases are operable. Sulfite Sulfite Sulfite as an intermediate. Reductases Questions the applicability of dsr or ‘ asr ’ the rhodanese reaction. 2 HS- Sulfide Investigating Enzymatic Pathways 4. Determine sulfide/thiosulfate ratio • 2:1 Both sulfur atoms of thiosulfate are reduced to sulfide • Strongly suggests the activity of a sulfite reductase 5. Genomic analysis Genome Results Anaerobic Genes for sulfite Rhodanese Rhodanese reductase Rhodanese dissimilatory sulfite reductase H. congolense were not Anaerobic retrieved. Molybdopterin sulfite protein Rhodanese reductase Molybdopterin H. vreelandii protein could potentially function as Molybdopterin protein Rhodanese Rhodanese thiosulfate reductase. H. kushneri Compiled Results H. congolense H. vreelandii H. kushneri H. salsuginis Thiosulfate -reduction Yes Yes Yes? No Rhodanese Activity Yes YesYes YesYes Yes Sulfide: Thiosulfate Ratio 2 to 1 2 to 1 1 to 1 NA Rhodanese, Rhodanese, thiosulfate Rhodanese, Predicted Genes sulfite reductase, thiosulfate NT reductase sulfite reductase reductase Investigating Enzymatic Pathways 2- 1 S2O3 Thiosulfate 5. Genome Analysis Thiosulfate Initial thiosulfate cleavage Rhodanese reductase remains unclear. rdl phs ° Can a rhodanese S-X* 2- SO3 cleave thiosulfate into ? Sulfite sulfite and sulfide? Anaerobic Suggests that an ‘anaerobic Sulfite sulfite reductase’ is involved - Reductase 2 HS ‘asr ’ in the metabolism of sulfite. Sulfide * Sulfur accepting molecule (traditionally CN in rhodanese) ‘Anaerobic sulfite reductase’ 2- • Also observed in SO3 (Inhibitory) Thermoanaerobacter , Clostridia and Sulfite Salmonella species • May be involved in reducing sulfite asrABC to prevent growth inhibition. • Is not amplifiable using gene targets for dissimilatory sulfite HS- reductase from SRB. Sulfide Rethinking TRB Gene Targets Rhodanese-like proteins are not practical gene targets for sulfidogenic Halanaerobium species. Anaerobic sulfite reductase is prime target Directly responsible for the production of sulfide. Inhibition of this enzyme be an effective means of control for sulfidogenic Halanaerobium species. Take Home Messages 2- 1 S2O3 Sulfidogenesis from Thiosulfate various sulfur anions and Rhodanese Thiosulfate alternative enzymatic rdl reductase pathways must be phs 2- considered. SO3 Anaerobic sulfite Sulfite reductase may be a more appropriate gene target Anaerobic for sulfidogenic Sulfite Halanaerobium species. Reductase 2 HS ‘asr ’ Sulfide Acknowledgements Thesis Committee: Dr. Kathleen Duncan Dr. Ralph Tanner Dr. Joseph Suflita Dr. Michael McInerney Thank you for your time. I welcome your questions!.