Phylogeny
Strain M-6
Salinity Measurements
Abstract
30
25
12
(A)
9
20
15
sulfur and sulfide and a stream salinity of 1%. We recently encountered an unexpected abundance of members of the
10
extreme halophilic Archaea of the order Halobacteraceae in the spring microbial mats. Measurement of the salinity at
Moisture (%)
moisture content compared to the values detected in the spring water, presumably as a result of evaporation. In this
A scanning electron micrograph of a exponential phase (Left) and
A stationary phase (right) culture of strain M6.
study we characterize one of the isolates, strain M6, as a new species. Strain M6 cells were pleomorphic and were
capable of growth at salt concentrations ranging from 6% to saturation, and at least 1 mM Mg2+ was required for
growth. Strain M6 was characterized by its ability to anaerobically reduce elemental sulfur to sulfide, and was
capable of growth in defined media with a range of carbon substrates including benzoate. 16S rRNA sequence
analysis indicated that it belongs to the family Halobacteriaceae, genus Haloferax, with similarity values between
96.7-98% to other described members of the genus, and 89% to Halogeometricum borinquense, its closest relative
outside the genus Haloferax. Polar lipid analysis further supported placement of strain M6 in the genus Haloferax.
Members of this genus are most commonly found in areas of high salt including salterns and the Dead Sea.
Salinity (--)
moisture content (-•-)
0
0
(B)
30
35
30
25
• Gram negative
25
20
• Motile
20
15
• Extremely pleomorphic
• Colonies were small (2-3 mm) and salmon pink in
color at 37°C
10
•16S rRNA sequence analysis of strain M6 showed a 96.7-98.0%
5
5
similarity to other validly described species of this genus
0
0
Biochemical and physiological characterization allowed differentiation of strain M6 from the other members of the
•M6 was 89% similar to Halogeometricum borinquense the most
genus Haloferax, and we propose a new species, Haloferax sulfurifontis, to accommodate this strain. This work
closely related species outside the genus
0
1
2
3
4
5
•Salt profiles revealed that there were
areas found throughout the mat and
on the banks where moisture
decreased and salinity increased
presumably due to evaporation.
•It is probable that these areas are
providing microenvironments that
facilitate the growth of these
Halophilic Archaea
Depth from surface (in cm intervals)
demonstrates that members of the Halobacteriaceae are not restricted to their typical hypersaline habitats, but can
also inhabit lower salinity environments where localized NaCl concentrations are high enough to allow their survival.
15
10
• Scanning Electron Microscopy revealed flat rods
and cocci
Core A: 5 cm from the stream bank
Core B: 30 cm from the stream bank
3
5
various locations of the spring and along vertical gradients revealed that shallow and superficial areas, where
microbial mats are usually encountered, have a much higher salinity (up to 32% NaCl concentration) and lower
6
Salinity (%)
Zodletone spring is located in Southwestern Oklahoma and is characterized by a high concentration of dissolved
Strain M6 can reduce S° anaerobically
Characterization
Sulfide production by H. sulfurifontis
Introduction
Temperature
optimum (0C)
Temperature range
(0C)
NaCl range (M)
NaCl optimum ( M)
Cell stability (M
NaCl)
Motility
pH optimum
Gelatin hyd rolysis
Starch hyd rolysis
Anaerobic nitrate
reduction
Tween 80
hyd rolysis
Indole production
H2S production
from thio sulfate
G+C content
(mol %)
Casein hyd rolysis
Resistance to
rifampi cin
Methods
Serially diluted (up to 10-7) mat material into a yeast extract
based halophilic medium(2) treated with kanamycin and
ampicillin (75µg/ml). Three different NaCl concentrations were
used (7,12 and 18%).
Zodletone Spring
Zodletone: Microbial Mats
Plated onto halophilic medium plates (1.5% agar).
Zodletone Spring
• An artesian sulfur containing, mesophilic spring located in
Strain
M6
32-37
H.
volcanii
40
H.
gibbonsii
35-40
H.
H.
H.
H.
denitrificans mediterranei alexandrinus lucentense
50
40
37
37
18-50
N.D.
25-55
30-55
20-55
20-55
10-45
1-5.2
2.1-2.6
0.5
1-4.5
1.7-2.5
0.5
1.5-5.2
2.5-4.3
0.5-0.7
1.5-4.5
2-3
1.5
1.3-4.7
2.9
0.5
1.8-5.1
4.3
1.7
1.8-5.1
4.3
ND
+
6.4-6.8
+
-
7
-
6.5-7
+
-
6-7
+
+
+
6.5
+
+
+
7.2
+
-
+
7.5
-
+
-
+
-
+
+
ND
+
+
+
+
+
+
+
+
-
+ (check)
+
+
+
60.5
63.4
61.8
64.2
59.1-62.2
59.5
64.5
+
-
+
-
-
+
-
+
ND
•Strain M-6 has a lower temperature optimum than other species of the genus Haloferax
•M-6 has a wide range of NaCl concentration in which it can grow
•M-6 can recover from low NaCl concentrations which could explain why it is able to survive in this low salt environment
•M-6 was capable of growth on a wide range of carbon substrates including benzoate
10mM) and S°. Short chain gaseaous alkanes (methane,
ethane and propane) impact the source water.
The plates were incubated at 37°C under light until pigmented
colonies began to grow (about 2 weeks)
• The spring salinity ranges from 0.7-1.0%
• Sunlight is an important energy source driving the formation
Growth and Survival of strain
M6 under Various NaCl
Concentrations
• The spring flows for approximately 20 meters before
discharging into a creek.
Individual colonies were picked from the plates, restreaked twice
and microscopically checked for purity.
Extreme Halophilic Archaea in Zodletone spring
(Family Halobacteriaceae)
• Molecular analysis of 16s rRNA of the spring community
indicated that 36% of Archaeal mat clones belonged to 5
different groups within the family Halobacteriaceae (1).
Growth rate (day-1)
of purple and green phototrophic mats throughout the spring.
2
1.6
1.2
0.8
Archaeal specific primers
5
10 15 20 25 30 35 40
Salt concentration (%)
1.0E+08
• Halobacteriaceae require high salt concentration ranging
from 3.5 M NaCl to saturation (5.2 M)
7.5E+07
The strain of interest was further characterized following the
guidelines provided by Oren et al. (2). All biochemical testing
followed procedures outlined in Reference (3).
Cells number (celss /ml)
• Members of this family are known to be aerobic
heterotrophs.
• Isolated from hypersaline environments (I.e. Salterns, the
Dead Sea, hypersaline lakes)
•When NaCl
concentrations are
as low as 4% M6
cells can maintain
their viablity
0.4
0
DNA was extracted from the colonies and PCR amplified using
Optimal NaCl
concentration for
growth of M6 is
12.5 - 15%
(--) distilled water (-o-) 1%
(--) 2%
(-∆-) 3%
(--) 4%
(-•-) 5%
(--) 15%
200
(-•-) Elemental sulfur
(-∆-) elemental sulfur, but no substrate
(-o-) Medium with no elemental sulfur
(--) Uninoculated medium
100
t0
0
0
10
20
4 days
30
Medium: Yeast extract, sulfur,
ferrous ammonium Sulfate
Time (Days)
•Interestingly, Strain M6 was capable of reducing sulfur under anaerobic conditions
•The abundance of this organism in this environment coupled with the ability of it to reduce elemental sulfur may
suggest that it has its’ own role in the sulfur cycle at Zodletone
Conclusions
•A novel species of the genus Haloferax was isolated from Zodletone Spring for which we propose the name
Haloferax sulfurifontis
•Characterization of strain M6 supported its’ affiliation with the genus Haloferax
•Closer analysis of the salt profiles of Zodletone indicated tha,t although overall spring salinity did not exceed 1%,
there are areas in the mat and on the banks where moisture decreases and the concentration of NaCl increases. These
hypersaline pockets may provide a suitable environment for the growth of extreme halophilic Archaea.
the Anadarko Basin of Southwestern Oklahoma
• Surface water contains high sulfide concentrations (8-
300
Sulfide produced (µM)
Characteristic
•Clone libraries indicate that there are diverse members of the halophilic Archaea present in the mat, however, only
one strain was successfully isolated
DNA-DNA
Hybridization
Source of
unlabelled DNA
G+C content
(mol%)
Strain M6
Haloferax volcanii
NCIMB 2012T
Haloferax
gibbonsii ATCC
33959T
Haloferax
denitrificans DSM
4425T
Haloferax
mediterranei
ATCC 33500T
Haloferax
lucentense JCM
9276T
60.5
63.4*
% Relatedness
with 3H labeled
DNA
100
21
61.8*
24
•This research proves that extreme halophilic Archaea may be more abundant in low salt environments than previously
believed if conditions allow for areas of high salt concentration to develop.
Current and Future Work
• Isolating novel halophilic Archaea using eleven different substrates and three different salt concentrations (18, 25
and 30%)
• Preliminary investigation suggest that two of the obtained isolates belong to novel genera within the family
Halobacteriales (only 92% and 93% similar to their closest cultured relatives).
•Few selected strains will be further characterized
64.2*
1
60.0*
4
64.5*
3
Table 2. DNA G+C con tents and leve ls of DNA-DNA hyb ridiz ation between strain M6 and
5.0E+07
References
other related halobacterial strains.
* Data taken from Mull akhanbh ai & Larsen (1975), Rodrigue z-Valera et al. (1983), Tomli nson
(1)
Elshahed, M.S., Najar, F.Z., Roe, B.A., Oren, A., Dewers, T.A. and Krumholz, L.R. (2004). Survey of archaeal diversity reveals an abundance of halophilic
Archaea in a low-salt, sulfide- and sulfur-rich sprin. Appl. Environ. Microbiol. 70: 2230-2239.
(2)
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriology 47, 233238.
(3)
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). Methods for General and Molecular Bacteriology, pp 227-248. Washington, D.C.: American
Society for Microbiology
et al. (1986), Jue z et al. (1986) and Gutierrez et al. (2002).
2.5E+07
• DNA-DNA hybridization studies showed that strain M6 had a hybridization
value between 0-24% to all other species of the genus Haloferax
0.0E+00
0
5
10
15
Time (days)
20
25