A novel lineage of sulfate-reducingmicroorganisms: Thermodesulfobiaceae
fam. nov., Thermodesulfobiumnarugense, gen. nov., sp. nov., a newthermophilic isolate from a hot spring
Koji Mori, Hongik Kim, Takeshi Kakegawa andSatoshi Handa
Extremophiles (2003) &: 283-290
Sulfate ReducingMicroorganisms
• 6 phylogenetic lineages• 4 Bacterial lineages in two
clades (δ-Proteobacteriaand Bacillus/Clostridiagroup)
• Bacterial lineages aremesophilic to moderatelythermophilic
• 2 Archaeal lineages in twogenera (Archeaoglobusand Caldivirga)
• Archaeal lineages arehyperthermophilic http://mcb.berkeley.edu/labs/kustu/mcb112/nov9.
htm
The Plan
• Isolate and characterize a novel thermophilicsulfate-reducer from Narugo hot spring in Miyagi,Japan
• Once isolated analyze 16S, DsrAB (dissimilatorysulfite reductase) and ApsA (alpha subunit of theadenosine-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 matwere inoculated into a reduced anaerobic mediumdesigned to enrich for sulfate reducers
• After 4 days of growth at 55°C pronounced H2Sproduction was detected
• After several transfers isolated colonies were seenon solid media
• Final purification on solid media yielded an isolateNa82T
Morphology and Physiology ofNa82T
• 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 ofNa82T
• pH requirements were between4-6.5
• Highest doubling time occurredbetween 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 otherbacterial sequences fromacross 21 phyla
• Used ARB software• NJ tree from this showed
that Na82T was divergentfrom most phyla (Seq.similarity less than 81%)
• Closest sequence wasfrom the candidate OP9division
*
Phylogenetic Analysis
• Used a subset of 16Ssequences from knownsulfate reducers wasused
• Strain Na82T treedwithin the sulfatereducing bacteria,however the bootstrapvalues were low *
Phylogenetic Analysis
• Carried out phlyogenetic analysis on deducedamino 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 werecreated (NJ tree was the starting point)
• NJ, ML and maximum parsimony (MP) treesshowed similar relationships
*
*
DsrAB phylogeny ApsA phylogeny
Conclusions
• Able to isolate an obligately anaerboic sulfatereducing bacteria from a hot spring near Narugo inMiyagi Prefecture, Japan
• Due to low sequence similarity in the 16S (lessthan 80%) it is proposed to be a novel lineage ofbacteria
• Thermodesulfobiaceae: Thermodesulfobium:Thermodesulfobium narugense
About Thermodesulfobiumnarugense
• 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 Thermodesulfobiumnarugense
• Sulfite, elemental sulfur, Fe(III), fumerate,dimethyl sulfoxide and O2 are not utilized aselectron acceptors
• Electron donors used in the presence of sulfate areH2 and formate
• Does not grow with glucose, acetate, lactate,pyruvate, malate, propionate, butyrate, fumerate,succinate, citrate, ethanol, propanol or methanol
FAME analysisSignificance & Origins
• Method of chemotaxonomy: classify organisms based onunique 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.
R–C –O–
=O glycerol
R–C –O–CH3
=Omethanolic HCl
The method of Mori et al.
Images from http://www.agilent.com
Some factors that impact anorganism’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) mayvary quantitatively (2)
• This is a qualitative technique, and one should onlyconsider the principle fatty acid component (1,2)
1) Mary Lechevalier. 1977. Crit. Rev. Microbiol. 5: 1092) Norman Shaw. 1974. Adv. Appl. Microbiol. 17: 63
Some factors that impact anorganism’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 performFAME 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’suse?
I want tolive forever
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