•16S rRNA gene analysis using bacterial primers identified several novel lineages in the spring community, Including members of at least 8 uncultured bacterial divisions (OD1, SR1, OP11, TM7, WW1, WS3, OP3, and ZB3), (Elshahed et al. 2003).

• A survey of the archaeal community also detected members of four yet-uncultured fungal lineages (UAG1, rice root cluster III , UAG3, and marine benthic group IV) (Elshahed et al. 2004a)

• while a survey of the micreukaryotic community detected a fungal (candidate phylum LKM) lineage (Luo et al. 2005).

Abstract

Zodletone spring in Southwestern Oklahoma is characterized by high dissolved sulfide and sulfur concentrations, presence of microbial mats, and short chain alkanes at the source of the spring. We are investigating the phylogenetic diversity of the microbial communities in the spring, as well as the unique metabolic capabilities of some of the identified microbial lineages that allow them to thrive in the sulfide-saturated spring sediments. Novel microbial lineages in Zodletone spring. Small subunit ribosomal gene-based analysis of the bacterial, archaeal, and eukaryotic communities in Zodletone spring indicated the presence of an extremely diverse microbial community. Of special interest was the detection of yet-uncultured bacterial (candidate divisions OD1, SR1, OP11, TM7, WWE1, WS3, and OP3) (Elshahed et al. 2003), archaeal (UAG1, rice root cluster III , UAG3, and marine benthic group IV) (Elshahed et al. 2004a), and fungal (candidate phylum LKM) lineages (Luo et al. 2005). Location and sequencing of a candidate division OD1 40 Kb fosmid indicated that this group has a low G+C content. Phylogenetic analysis of several OD1 genes confirmed the status of this group as a novel bacterial division. The presence of genes indicative of anaerobic metabolism is consistent with the fact that this group has mainly been encountered in anaerobic habitats (Elshahed et al. 2005).

Halophilic Archaea in Zodletone spring. Survey of archaeal diversity in the spring revealed the abundance of halophilic Archaea, especially in the variably hypersaline soils adjacent to the spring (Elshahed et al. 2004a). We isolated several halophilic Archaea from the spring, and these isolates represent 3 novel genera within the order Halobacteriales (Haladaptatus, Halosarcina, and Halomultiformis), and one new species, Haloferax sulfurifontis (Elshahed et al. 2004b, Savage et al. 2007, Savage et al. in preparation). Physiological studies have shown that strains from the spring are able to tolerate relatively low salt levels and many can respire elemental sulfur. Both of these characteristics may be adaptations to the spring environment. Additional evidence for this interpretation comes from the fact that isolates related to the genera, Haladaptatus and Halomultiformis have so far only been identified in relatively low salt habitats.

Anaerobic heterotrophic Planctomycetes in Zodletone spring. The mechanism of survival of heterotrophic Planctomycetes in anaerobic habitats is yet unclear. Culture-independent analysis indicated that an extremely diverse community of Planctomycetes is present in spring sediments. Using complex organic media we isolated a novel strain that belongs to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. Detailed characterization of this isolate revealed its ability to anaerobically reduce elemental sulfur to sulfide, as well as to produce acids from sugars (Elshahed et al., In preparation). Both characteristics could potentially be adaptations to the anaerobic, sulfide, and sulfur-rich environment at the spring source.

Biodegradation of short chain (C2-C6) n-alkanes under methanogenic and sulfate-reducing conditions. To investigate the ability of the native microbiota to metabolize the abundant short chain hydrocarbons (C2-C6) present in the spring, enrichments were set up using source sediments under sulfate-reducing and methanogenic conditions. Under sulfate-reducing conditions, propane, pentane and hexane were degraded, and these capabilities were maintained upon subculturing. Analysis of 13C values of residual propane in active enrichment showed that it became depleted in 12C, confirming the fact that biodegradation is occurring. Under methanogenic conditions, ethane was observed to be degraded.

Zodletone Spring

• An artesian sulfur-containing, mesophilic spring located in the Anadarko Basin in Southwestern Oklahoma.

• Surface water contains abundant sulfide (8-10 mM) and zero valent sulfur (1-1.5 mM), as well as methane, ethane, and propane.

• The source is a contained area, approximately 1 m2, filled with biomass and soft sediments to a depth of at least 15 cm

• High light exposure results in the abundance of microbial mats through the course of the spring.

• The spring flows for approx. 20 meters before discharging into stinking creek.

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Overview of Zodletone spring

Source of the spring Microbial mats

Goals of the Zodletone Spring Microbial Observatory1. Investigate the diversity of the bacterial, archaeal, and

microeukaryotic community in the spring using phylogenetic, metagenomic, and isolation procedures, with special emphasis on members of yet-uncultured bacterial lineages.

2. Investigate the metabolic capabilities of specific microbial lineages that were identified in the spring but are not usually encountered in anaerobic sulfide-saturated habitats, in order to identify their mechanisms of growth and growth in the strictly anaerobic, sulfide-saturated sediments of the spring.

Metagenomic analysis of the microbial community in Zodletone Spring

• A large insert library of 19,200 fosmids was constructed from Zodletone spring sediments.

• The library was screened for inserts containing 16S rRNA gene as a phylogenetic anchor, 36 fosmids with 16S gene were detected.

• One fosmid (ZFos45e05) belonging to candidate division OD1 was located and sequenced (Elshahed et al. 2005)

General characteristics of ZFos45e05

• Low G+C content (34.9)• rRNA operon organization in which 5, 16, and 23S rRNA genes are separated• High percentage and hypothetical and conserved hypothetical proteins

ZFos45e05

Length (bp) 35743 G+C% 34.9

G+C% (coding regions) 40.4 rRNA operon 16S

Number of ORFs1 33

Number of tRNA 13

Hypothetical proteins (unique) 13 Conserved hypothetical proteins (hits

to othrer hypothetical proteins) 3

Phylogenetic affiliation of most similar orthologs

Proteobacteria 5 Firmicutes 3

Aquifex 1 Fusobacteria 1

Actinobacteria 1 Chloroflexi 1

Archaea 5 Eukaryotes 3

• Phylogenetic analysis of three putative housekeeping genes in ZFos45e05 (DNA polymerase 1 (ORF 1), tRNA (guanine-N1)-methyltransferase (ORF 10), and large ribosomal subunit protein L19 (ORF 32) supported the hypothesis that candidate division OD1 is not closely related to any bacterial division with genome-sequenced representatives.

• The large sequence divergence which resulted in deep branching points to the potential genomic novelty of this yet-uncultured phylum of Bacteria.

A. DNA polymeraseB. tRNA (guanine-

N1)-methyltransferase

C. large ribosomal subunit protein L19

Novel microbial lineages in Zodletone Spring

Novel Halophilic Archaea from Zodletone Spring

In a 16S rRNA gene analysis of the archeal community in the spring, 36% of the mat and 4% of the source clones belonged to the order Halobacteriales, members of which are known to grow in aerobic, high salt environments (Elshahed et al. 2004a).

Depth from surface (in cm intervals)05101520253035

051015202530

0 1 2 3 4 5

Moisture (%)

0

3

6

9

Salinity (%)

0510152025 (A)

(B)

Measurement of the salinity at various locations at the spring, as well as along vertical gradients revealed that shallow and surficial areas where microbial mats are usually encountered have a much higher salinity (up to 32% NaCl concentration), and lower moisture content compared to the values detected in the spring water, presumably from evaporation

Salinity moisture content

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(1)

(2)

(2)

(3) (4)

Using a high-salt plus antibiotic media with twelve different substrates, 35 different strains of halophilicArchaea were isolated. These strains belonged to 4 different groups: Members of the genus Haloferax (1), and members of three novel archaeal genera: Haladaptatuus (2), Halosarcina (3), and Halomultiformis (4) (Elshahed et al. 2004b, Savage et al. 2007, Savage et al., In preparation)

Adaptations of Zodletone halophilic Archaea to low salt and low oxygen conditions

Haloferax sulfurifontis can grow under anaerobic conditions, reducing elemental sulfur to sulfide

Sulfide produced (µM)

0

100

200

300

0 10 20 30

Time (Days)

Medium: Yeast extract, sulfur, ferrous

ammonium Sulfate,

Cells of Haladaptatus paucihalophilus remained viable after prolonged exposure to distilled water.

%NaCl 0 4 12 24 48 168*

0 + + + + + 2.4x109

1.0 + + + + + 2.4x109

2.0 + + + + + 2.4x109

3.0 + + + + + 9.3x108

18.0 + + + + + 9.3x108

*Number of cells (cells/ml) recovered after 1 week. Determined by diluting cells from 10-1 to 10-12 in triplicate.

Phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in Zodletone spring•Planctomycetes have been often detected

in a variety of anaerobic environments, including Zodletone spring.

•No anaerobic hetertrophic planctomycete haveyet been isolated.

•The Planctomycetes community at the spring was studied using a combination of culture-independent analysis and isolation methods.An extremely diverse Planctomycetes community thrives in the anaerobic sediments at the spring source

•Culture-independent analysis of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring (Elshahed et al. In preparation).

•Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, yet-uncultured Planctomycetes lineages.

Isolation of an acid-producing, sulfur-reducing Planctomycete from source sediments•Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellua-Blastopirellula clade within the Planctomycetes.•The closest validly described culture relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity).•Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars, both characteristics could potentially

allow strain Zi62 to survive and grow in the anaerobic, sulfide, and sulfur-rich environment at the spring source. Sulfide production by isolate Zi62 when inoculated in anaerobic medium supplemented with (- �-) Elemental sulfur, (--) elemental sulfur, but no substrate (0.1% sucrose) included in the medium, (--) inoculated medium with sulfur, but no substrate, (--) Uninoculated medium with elemental sulfur but no substrate. All values shown

are average of triplicate tubes.

Product mols produced or utilized mols produced / 100 mol sucrose1 % substrate recovered as Sucrose 79.29 NA NA Succinate 64.2 80.97 26.99 Acetate 126.3 159.29 26.55 Lactate 18.45 23.27 5.82 Propionate 12.3 15.15 3.88 Formate 6.6 8.23 0.86 CO2 400.7 505.54 42.11 % C. Recovery 106.0

Sugar fermentation by strain Zi62

Anaerobic degradation of short cain hydrocarbons in Zodletone spring

• Short-chain n-alkanes are abundant at the spring source (methane, ethane and propane)• To enrich for organisms capable of degrading short-chain alkanes, source sediment and

groundwater was amended with a hydrocarbon (C2-C6) and incubated under different electron-accepting conditions

• Under sulfate-reducing conditions propane, pentane and hexane have been degraded.• A loss of ethane and propane was observed under methanogenic conditions.• All sulfate-reducing enrichments have been successfully subcultured and maintained

Sa m p l e C3

H8

CO2

C3

H8

(S u bs t rate added) - 22.7 (±0.9)

C3

H8

in St e r il e - C on tr ol - 23.2 (±0.8) - 10.6 (±0.1)

C3

H8

in act iv e bott l es in l a g p h ase - 22.6 - 13.8

Res i d u a l C3

H8

in or igin a l e n r i c hm e n ts

a f ter >90% de g rad a t i o n

- 20.6 (±1.3) - 9.8 (±0.1)

Res i d u a l C 3 H 8 in s u bc ul t u red

e n r i c h m e n ts a f ter >9 0 % de g radat i o n

- 16.0 (±2.0) - 16.8 (±0.6)

Alka n e Sul f a t e - R edu c ing M e thanoge n ic

Ethane - + (1100)

Propane ++ (485) + (1100)

Buta n e - -

Pen t ane ++ (207) -

Hexa n e ++ (600) -

(++) Loss of >60% of substrate, with little to no loss in the sterile-control. (+) Loss of 40-60% of substrate, with little to no loss in the sterile-control. (-) Loss of <40% of substrate. (+*) methane detected

Metabolism of C2-C6 n-alkanes under sulfate-reducing and methanogenic conditions. Days at which measurements were taken are indicated in parenthesis

Analysis of 13C (%) values of residual propane in active enrichment showed that it became depleted in 12C by 6.7% (2.0%) after 660 days of incubation.

Conclusions

•An extremely diverse microbial community is present in Zodletone spring, and includes at least 7 bacterial, 4 archaeal, and 1 fungal candidate phyla•A 35.6 KB OD1 genomic fragment was located and sequenced. The fragment has a low G+C content, and an interesting rRNA operon arrangement, and contained general genes suggestive of anaerobic fermentative metabolism.•A diverse community of halophilic Aarchaea (order Halobacteriales) was detected in the spring. Characterization of several Halobacteriales isolates suggest that this group of aerobic obligate halophiles can survive in Zodletone spring by having a relatively low salt requirement for survival and/or a capability to grow anaerobically by respiring elemental sulfur to sulfide•Members of all major lineages of the Planctomycetes are present in the anaerobic, sulfide-saturated spring source. Sulfur reduction and mixed acid fermentation represent two of the possible mechanisms used by heterotrophic Planctomycetes to grow in anaerobic environments.•Zodletone spring native microbiota possess the capability to degrade propane, n-pentane, and n-hexane under sulfate reducing conditions, and ethane under

methanogenic conditions.Publications from the Zodletone spring microbial observatory

1. Elshahed MS, JM Senko, FZ Najar, SM Kenton, BA Roe, TA Dewers, JR Spear, and LR Krumholz 2003. Bacterial diversity and sulfur cycling in a mesophilic sulfide-rich spring. Appl. Environ. Microbiol. 69:5609-5621.2. Elshahed MS, FZ Najar, BA Roe, A Oren, TA Dewers, and LR Krumholz 2004. Survey of archaeal diversity reveals abundance of halophilic Archaea in a low-salt, anaerobic sulfur spring. Appl. Environ. Microbiol. 70:2230-2239.3. Senko JM, BS Campbell, JR Henricksen, MS Elshahed, TA Dewers, and LR Krumholz 2004. Barite deposition mediated by phototrophic sulfide oxidizing bacteria. Geochem. Cosmochem. Acta. 68:773-780.4. Elshahed MS, KN Savage, A Ventosa, M Carmen-Gutierrez, A Oren, and LR Krumholz 2004. Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide and sulfur-rich spring. Int. J. Syst. Evol. Microbiol. 54:2275-2279.5. Luo Q, LR Krumholz, F Z Najar, BA Roe, AD Peacock, DC White, and MS Elshahed 2005. Diversity of the microeukaryotic community in sulfide-rich Zodletone spring. Appl. Environ. Microbiol. 71: 6175-6184.6. Elshahed MS, FZ Najar, M Aycock, C. Qu, BA Roe, and LR Krumholz 2005. Metagenomic analysis of the microbial community at Zodletone spring: Insights into the genome of novel Candidate division OD1. Appl. Environ. Microbiol. 71: 7958-7962.7. Savage KN, LR Krumholz, A Oren and MS. Elshahed 2007. Haladaptatus paucihalophilus gen. nov., sp. nov., a Halophilic Archaeon Isolated from a Low-Salt, High-Sulfide Spring. Int. J. Syst. Evol. Microbiol. 57:19-24.Manuscripts in preparation8. Savage KN, LR Krumholz, A Oren and MS. Elshahed. Halosarcina Oklahomense gen. nov., sp. nov., a halophilic Archaeon Isolated from Zodletone spring in southwestern Oklahoma. Int. J. Syst. Evol. Microbiol.9. Elshahed MS, Sisk TM, FZ Najar, Q Luo, BA Roe, SM Buehring, and LR Krumholz. Diversity of members of the phylum Planctomycetes and description of Cartipirellula succinogenes gen. nov. sp. nov. a succinate-producing, sulfur-reducing Planctomycete from Zodletone spring (OK). Appl. Environ. Microbiol.

Zodletone Spring Microbial Observatory: Phylogenetic Diversity and Metabolic Capabilities of Novel Microorganisms In Sulfide-Saturated Habitats

Mostafa S. Elshahed (P.I.)1 , Lee R. Krumholz (P.I.)2, Stephan Sievert3(P.I.), Kristen N. Savage2, and Paul Philp2

1Oklahoma State University, Stillwater, OK, 2University of Oklahoma, Norman, OK, and 3 Woods Hole Oceanographic Institute, Woods Hole, MA