Transcript Conference16 - Zodletone Spring

Halophilic Archaea at
Zodletone Spring
Kristen Savage
Zodletone Geochemistry
Source
•High concentrations
of dissolved S2(8-10mM)
•0.2 M NaCl
•Barium and sulfate
•Dissolved Sulfur
•Barite and calcite
Archaeal Diversity
Source Archaea
Crenarcharcheota
6%
UAG
36%
Methanogens
54%
Halophiles
4%
Mat Archaea
Crenarchaeota
12%
Uncultured
6%
Halophiles
36%
Methanogens
46%
Phylogeny of
Halophilic Archaea
• 5 different groups within
the order Halobacteriales
• Groups I & II represent
novel genera
• Groups III & V related to
previously described
genera
• Group IV represented all
source clones
(Elshahed et al. 2004)
Halophilic
Archaea
http://141.150.157.117:8080/prokPUB/index.htm
• Dominate hypersaline
environments
• Require at least 8% NaCl for
growth (20-26% NaCl)
• Aerobic
• Heterotrophic
• Characterized by red, pink or
orange coloration
• Order Halobacteriales
– 15 genera, 44 species
• Variety of shapes and sizes
Spring Salinity
30
25
9
20
Moisture (%)
15
6
10
3
5
0
30
0
35
(B)
30
25
25
20
20
15
15
10
(Elshahed et al. 2004)
Salinity (--)
5
moisture content (-•-)
0
Salinity (%)
• Stream Salinity 0.7-1.0%
• Mat Salinity 2-5%
• Soil (top layer)
• 5 cm- >30%
• 30 cm- >25%
12
(A)
10
5
0
1
2
3
4
5
0
Depth from surface (in cm intervals)
A Continued Search for
Halophiles
• Clone libraries indicated the presence of a diverse and
novel halophilic community
• Originally18 strains were isolated from the mats present at
the stream
• Studies indicated that these isolates were of the same
species
• In order to stimulate the growth of different isolates the
medium was prepared with 3 different salt concentrations
(18, 25 and 30%) and 11 different carbon sources.
Ampicillin and Kanamycin were used to select against
halotolerant bacteria
Preliminary Screening
• Sequenced approximately 30 isolates
• Isolates clustered into four different groups
• Two groups (III and IV) clustered with
previously defined genera
(Halogeometricum and Haloferax)
• Groups I and II represented novel genera,
but clustered closely with some uncultured
clones
Diversity of
Halophilic
Isolates
• BZ256 is 98% similar to
ZAR25 (Clone Group III)
• Novel Group II isolates
cluster with ZAR31(Clone
Group I)
• Clone groups II, IV, and V
had no cultured
representatives
Novel Group II
NaCl Range (optimum)
MgCl2
pH Range (optimum)
Temperature Range
(opt.)
Colony Morphology
Cell Morphology
Doubli ng Time
% Relatedness
DX253
5-30% (18%)
5-200 mM
4.5-7.5 (6.5)
25-45C (30C)
GY252
5-30% (15%)
5-200 mM
4.5-9 (6.0)
25-45C (25C)
Large round co lonies,
raised, transparent, dark
pink
Medium cocci, tetrads and
clumps
13.25 hr/gen
92% Natronorub rum
tibetense
Small round colon ies,
raised, transparent, light
pink
Smaller cocci, singles and
pairs
8.61 hr/gen
89% Natronorub rum
tibetense
Sugars
0.8
O.D. = 600nm
Sugar
Alcohol
0.7
0.6
Organic
Acids
0.5
0.4
Amino
Acids
0.3
0.2
PY
MAN
SO
GYR
MAN
SO
GYR
SNT
SNT
PY
FA
CT
FA
CT
LT
MA
MA
AC
LT
GAL
FS
LAC
ST
XY
DX
SUC
AA
GN
AL
GLT
AG
Positive
0
Negative
0.1
Substrate
GY252
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Substrate
AC
GAL
LAC
FS
ST
DX
XY
SUC
AA
GN
GLT
AL
AG
Positive
0
Negative
• Poor growth on amino
acids
• Preferred substrates:
Glucose, sucrose and
starch
• GY252 had a larger
range of substrate
utilization
0.9
O.D. = 600nm
Metabolism
Group II
DX253
Viability and Recovery
• Determine the ability of cells to recover
from low salt conditions
• Prepared HM (0-5%) NaCl and sterile H2O
solutions (0-5%) NaCl
• Inoculated washed cells and attempted to
recover the cells in standard HM liquid at
various time points
Viability and Recovery
DX253
Sterile H2O
Halophilic
Medium
0%
0.5%
1.0%
2.0%
3.0%
4.0%
5.0%
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Conclusions
• Haloarchaea at Zodletone Spring are numerous,
diverse and culturable
• Extreme halophiles may be more ubiquitous in
nature than previously believed
• May play an important role is sulfur cycling at
Zodletone and may be essential to other cycling
processes in other environments
Acknowledgements
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Dr. Krumholz
Dr. Elshahed
Dr. Oren
Dr. Ventosa
Tracy Sisk