Haloferax sulfurifontis sp. nov., a Halophilic Archaeon Isolated from

a Low Salt Sulfide-rich Spring

K. N. Savage1, M. S. Elshahed1, A. Oren2, L. R. Krumholz1

1University of Oklahoma, Norman, OK, 2The Hebrew University of Jerusalem, Jerusalem,

ISRAEL

Zodletone Spring

http://fermi.jhuapl.edu/states/maps1/ok.gif

• 8 l•min-1

• 20 m from source to creek

• High sulfide concentrations (8-10mM)

• 0.2 M NaCl• F, Br, Li, B, Sr, Ba,

SO42-

• BaSO4 and CaCO3

• Dissolved sulfur

Archaeal Diversity

Source Archaea

Methanogens54%

Halophiles4%

UAG36%

Crenarcheota6%

Mat Archaea

Methanogens46%

Halophiles36%

UAG 6%

Crenarchaeota12%

Halophilic Archaea

• 14 validly described genera• Isolated from hypersaline environments (i.e. The

Dead Sea)• Rods, cocci and pleomorphic forms• Require high salt concentrations from 3.5 M NaCl

to saturation (5.2 M)• Contain pigment material (bacteriorhodopsin and

halorhodopsin) which gives them a distinct pink or red color

Isolation of Haloarchaea

• 36% of the mat and 4% of the source library• Isolated halophiles from the mat using a

high salt plus antibiotic medium with different salt concentrations (7, 12 and 18%)

• Serially diluted mat material and plated onto HM plates (immediately and after incubation)

• Cultures were incubated at 37ºC under light

A Novel Species of Haloferax

• 16S rRNA sequence analysis of strain M6 showed a 96.7-98.0% similarity to other validly described species of this genus

• M6 was 89% similar to Halogeometricum borinquense the most closely related species outside the genus

• DNA-DNA hybridization confirmed its novel speciation having a hybridization value of only 24% to Haloferax gibbonsii

G+C content % Relatedness with 3H-labelled DNA

(mol%) from strain M6

Source of unlabelled DNA

_________________________________________________________________________________

___

Strain M6 60.5 100

Haloferax volcanii NCIMB 2012T 63.4* 21

Haloferax gibbonsii ATCC 33959T 61.8* 24

Haloferax denitrificans DSM 4425T 64.2* 1

Haloferax mediterranei ATCC 33500T 60.0* 4

Haloferax lucentense JCM 9276T 64.5* 3

* Data taken from Mullakhanbhai & Larsen (1975), Rodriguez-Valera et al. (1983), Tomlinson et al.

(1986), Juez et al. (1986) and Gutierrez et al. (2002).

Phylogeny

Haloferax sulfurifontis• Isolated from Zodletone

spring• Extreme pleomorphism• Colonies were small (2-3

mm) and salmon pink in color at 37°C

• Growth occurred in a wide range of salt concentrations (6% to saturation)

• Required at least 1 mM Mg2+ for growth

• Anaerobically reduces Sº

Haloferax sulfurifontis

Characteristic Strain M6 H. volcanii H. gibbonsii H.denitrificans

H.mediterranei

H.alexandrinus

H.lucentense

Temperature optimum(0C)

32-37 40 35-40 50 40 37 37

Temperature range (0C) 18-50 N.D. 25-55 30-55 20-55 20-55 10-45NaCl range (M) 1-5.2 1-4.5 1.5-5.2 1.5-4.5 1.3-4.7 1.8-5.1 1.8-5.1

NaCl optimum (M) 2.1-2.6 1.7-2.5 2.5-4.3 2-3 2.9 4.3 4.3Cell stability (M NaCl) 0.5 0.5 0.5-0.7 1.5 0.5 1.7 ND

Motility + - - - + - +pH optimum 6.4-6.8 7 6.5-7 6-7 6.5 7.2 7.5

Gelatin hydrolysis + - + + + + -Starch hydrolysis - - - - + - -Anaerobic nitrate

reduction- - - + + - -

Tween 80 hydrolysis + - + - + + NDIndole production + + + - + + (check) +

H2S production fromthiosulfate

+ + + + - + +

G+C content (mol%) 60.5 63.4 61.8 64.2 59.1-62.2 59.5 64.5Casein hydrolysis - - + - + - -

Resistance to rifampicin + - - - - + ND

A Continued Search for Halophiles

• Clone libraries indicated the presence of a diverse 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 encourage the growth of different isolates the media was prepared at 3 different salt concentrations (18, 25 and 30%) and 11 different carbon sources were used instead of yeast extract

Future Work

• Microscopic inspection of colonies to determine cell morphologies present and trends

• Further purification and isolation

• Colony PCR and sequencing

• Anaerobic reduction of S°

• Strain characterizations

Acknowledgements

• Dr. Krumholz• Dr. Elshahed• Aharon Oren and Antonio Ventosa• Roe Laboratory

Funding for this project is by the National Science Foundation