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
http://mcb.berkeley.edu/labs/kustu/mcb112/nov9.
hyperthermophilic
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
T
Na82
•
•
•
•
•
•
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
T
Na82
•
•
•
•
•
•
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 5055ºC
• 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.
methanolic HCl
O
=
R–C –O–
glycerol
=
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