A novel lineage of sulfate-reducing microorganisms: Thermodesulfobiaceae fam. nov., Thermodesulfobium narugense, gen. nov., sp. nov., a new thermophilic isolate from a hot spring Koji Mori, Hongik Kim, Takeshi Kakegawa and Satoshi Handa Extremophiles (2003) &: 283-290 Sulfate Reducing Microorganisms • 6 phylogenetic lineages • 4 Bacterial lineages in two clades (δ-Proteobacteria and Bacillus/Clostridia group) • Bacterial lineages are mesophilic to moderately thermophilic • 2 Archaeal lineages in two genera (Archeaoglobus and Caldivirga) • Archaeal lineages are hyperthermophilic http://mcb.berkeley.edu/labs/kustu/mcb112/nov9. htm The Plan • Isolate and characterize a novel thermophilic sulfate-reducer from Narugo hot spring in Miyagi, Japan • Once isolated analyze 16S, DsrAB (dissimilatory sulfite reductase) and ApsA (alpha subunit of the adenosine-5’-phosphosulfate reductase) • Characterize cellular morphology • Characterize fatty acid content and genomic G+C content Miyagi Prefecture, Japan Growth and Isolation • Isolated sections of the collected microbial mat were inoculated into a reduced anaerobic medium designed to enrich for sulfate reducers • After 4 days of growth at 55°C pronounced H2S production was detected • After several transfers isolated colonies were seen on solid media • Final purification on solid media yielded an isolate Na82T Morphology and Physiology of Na82T • Rod-shaped (0.5x2-4 µm) • Non-motile • Non-spore forming • Gram negative • Strict Anaerobe • Growth coupled to Sulfate reduction • Temperature regieme between 37-65°C • Optimum 50-55°C Morphology and Physiology of Na82T • pH requirements were between 4-6.5 • Highest doubling time occurred between pH of 5.5-6.0 • Genomic G+C content is 35.1% • Menaquinone (MK)-7(H2) and MK-7 were the major fractions • MK-7(H4) and MK-8 were minor fractions • C16:0 was the major fraction of cellular fatty acids (45.7%) Phylogenetic Analysis * • 16S rRNA from Na82T was analyzed (1363 bp) (8F/1491R) • Compared to 350 other bacterial sequences from across 21 phyla • Used ARB software • NJ tree from this showed that Na82T was divergent from most phyla (Seq. similarity less than 81%) • Closest sequence was from the candidate OP9 division Phylogenetic Analysis • Used a subset of 16S sequences from known sulfate reducers was used • Strain Na82T treed within the sulfate reducing bacteria, however the bootstrap values were low * Phylogenetic Analysis • Carried out phlyogenetic analysis on deduced amino acid sequences of both DsrAB (dissimilatory sulfate reductase) and ApsA (adenosine-5’-phosphosulfate reductase) • Aligned deduced sequences with CLUSTALW • Created a neighbor-joining (NJ) tree, and then a maximum-likelihood (ML) matrix and tree were created (NJ tree was the starting point) • NJ, ML and maximum parsimony (MP) trees showed similar relationships * * DsrAB phylogeny ApsA phylogeny Conclusions • Able to isolate an obligately anaerboic sulfate reducing bacteria from a hot spring near Narugo in Miyagi Prefecture, Japan • Due to low sequence similarity in the 16S (less than 80%) it is proposed to be a novel lineage of bacteria • Thermodesulfobiaceae: Thermodesulfobium: Thermodesulfobium narugense About Thermodesulfobium narugense • Rod shaped, 0.5um X 2-4um in length • Non-motile and non-spore forming • Growth between 37-65ºC with an optimum of 50- 55ºC • pH range was between 4.0-6.5 • No growth occurs with a NaCl conc. above 1% (w/v) • Doubling time is 14h under optimum conditions • Sulfate, thiosulfate, nitrate and nitrite are used as electron acceptors About Thermodesulfobium narugense • Sulfite, elemental sulfur, Fe(III), fumerate, dimethyl sulfoxide and O2 are not utilized as electron acceptors • Electron donors used in the presence of sulfate are H2 and formate • Does not grow with glucose, acetate, lactate, pyruvate, malate, propionate, butyrate, fumerate, succinate, citrate, ethanol, propanol or methanol FAME analysis Significance & Origins • Method of chemotaxonomy: classify organisms based on unique fatty acid profiles • Why chemotaxonomy and why fatty acids? • Early 1960’s: physical and biochemical traits • Chemotaxonomy increased the arsenal • Fatty acids are relatively quickly and easily isolated, analyzed and identified Abel, K., H. de Schmertzing, and J.I. Peterson. 1963. J. Bacteriol. 85: 1039-1044 The method of Mori et al. O= O= glycerol methanolic HCl R–C –O– R–C –O–CH3 The method of Mori et al. Images from http://www.agilent.com Some factors that impact an organism’s fatty acid profile • Growth: temperature, pH, media composition (1) • Time of harvesting (1) • Different extraction procedures (1) • Preparations in different labs (or even the same lab) may vary quantitatively (2) • This is a qualitative technique, and one should only consider the principle fatty acid component (1,2) 1) Mary Lechevalier. 1977. Crit. Rev. Microbiol. 5: 109 2) Norman Shaw. 1974. Adv. Appl. Microbiol. 17: 63 Some factors that impact an organism’s fatty acid profile Abel, K., H. de Schmertzing, and J.I. Peterson. 1963. J. Bacteriol. 85: 1039-1044 Concluding statement • Standardized conditions are vital to proper identification, but…different bugs = different media • To what level can taxonomy be assigned? • What else can FAME tell us: • changes in community structure (phylum or broader) over time (1,2) • There are at least 2 (sister) companies that will perform FAME analysis: • Microbial Identification Inc. (MIDI) and Microbial ID 1) Haak, S.K., H. Garchow, H., D.A. Odelson, L.J. Forney and M.J. Klug. 1994. Appl. Environ. Microbiol. 60: 2483 2) Steger, K., A. Jarvis, S. Smars, and I. Sundh. 2003. J. Microbiol. Meth. 55: 371 Haak, S.K., H. Garchow, H., D.A. Odelson, L.J. Forney and M.J. Klug. 1994. Appl. Environ. Microbiol. 60: 2483 FAME - has it outlived it’s use? I want to live forever.