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