Oklahoma State University Department of Microbiology and Molecular Genetics 315 LSE building

1. (a) CM (b) Pa n P Funding Provided By National Science Foundation Microbial Observatories Program Introduction • Phylum Planctomycetes • First described based on microscopic observation in 1924, first pure culture representative obtained in pure culture in 1973 in dilute organic media (1). • Planctomycetes strains had been isolated from a variety of marine, and freshwater environments, such as marine brackish sediments, freshwater sediments, tissues of marine organisms, hot springs, and soil (2-6) • 16S rRNA gene-based surveys suggest that the Planctomycetes are ubiquitous in aerobic and anaerobic habitats, and that novel, yet uncultured lineages within the group exists. • Of special interest is the phylogeny and metabolic capabilities of members of the heterotrophic Planctomycetes in anaerobic habitats, since all described heterotrophic Planctomycetes are aerobic, chemoroganotrophic • 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. • The spring flows for approx. 20 meters before discharging into stinking creek. • 16S rRNA gene analysis using Bacteria specific primers identified Planctomycetes as members of the bacterial community at the spring source Isolation of heterotrophic Planctomycetes from Zodletone spring Two isolates, were obtained on plates containing either source sediment aqueous extracts or 5% rumen fluid as a sole carbon source. Strain Zi62 was successfully purified by re-streaking on media with N-acetylglucosamine as the sole carbon and nitrogen source, and with ampicillin and cycloheximide (50 g/ml each) to inhibit the growth of other Bacteria and Fungi. Zi62 cells are ovoid to pear-shaped, 0.6-1.2µm x 1.6-2.2µm, and occur in singles, pairs or in rosette formations attached at the smaller cell pole. Negatively stained cells showed that, similar to other Planctomycetes, crateriform-like structures are present, occurs only at one pole of the cell, and covers one-fourth to one-third of the cell surface. Thin-sectioned cells showed the presence of an intracellular membrane (ICM), dividing the cell into a pirellulosome and the paryphoplasm. A dense nucleoid structure was also observed that covered 20-30% of the pirellulosome. Overview of Zodletone spring Source of the spring A. Negative stained electron micrograph of strain Zi62, showing crateriform like structures (CM) covering one third of the cell, and a single flagellum. B. Thin sectioned micrograph of strain Zi62 showing the pirellulosome (Pl), the paryphoplasm (Pa), and the nucleoid structure (n). 0.05 substitutions/site Phylogenetic and metabolic diversity of the phylum Planctomycetes from an anaerobic, sulfide and sulfur-rich spring (Zodletone Spring, OK, USA) Mostafa S. Elshahed1, Noha H. Youssef1, Qingwei Luo2, Fares Z. Najar2, Bruce A. Roe2, Tracy M. Sisk2,Solveig M. Buehring3, Kai-Uwe Hinrichs3, and Lee R. Krumholz2 1Oklahoma State University, Stillwater, OK, 2University of Oklahoma, Norman, OK, and 3University of Bremen, Germany Oklahoma State University Department of Microbiology and Molecular Genetics 315 LSE building Stillwater, OK 74078 Mostafa@Okstate.edu N 147 • Abstract • We investigated the phylogenetic diversity and metabolic capabilities of the phylum Planctomycetes in the anaerobic sediments of a mesophilic spring (Zodletone spring) in southwestern Oklahoma. • Culture-independent analysis of 16S rRNA gene sequences suggested that an extremely diverse community of Planctomycetes is present at the spring. 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. • Using complex organic media, we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. Both strains had an identical 16S rRNA gene sequence, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou beach (Hawaii), a marine aquarium, and tissues of marine organisms (Aplysina sponge and post larvae of the giant tiger prawn Penaeus monodon). The closest validly described cultured relative of these strains was Blastopirellula marina (93.9% sequence similarity). • Detailed characterization of one of the isolates (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. • Based on the geochemical characteristics of this site, we have concluded that the capability of anaerobic metabolism is widely distributed among all major Planctomycetes lineages. Carbohydrate fermentation and sulfur reduction are possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions. Potential adaptations of strain Zi62 to anaerobic environments Culture-independent analysis of the Planctomycetes community at Zodletone spring source sediments Plot of sulfide production over time in anaerobic medium (--) inoculated, with elemental sulfur and substrate (sucrose 0.1%) added, (--) inoculated, with elemental sulfur but no substrate, (--) uninoculated, with elemental sulfur and substrate (--) Uninoculated,with no elemental sulfur and no substrate. All values shown are average of triplicate tubes. • 1. Anaerobic reduction of elemental sulfur • Strain Zi62 reduced elemental sulfur to sulfide under anaerobic conditions, and the production of sulfide was coupled to the disappearance of the sulfur precipitate in active incubations. • Anaerobic sulfur respiration is mediated by polysulfide reductase, which catalyzes the reduction of polysulfide to sulfide. Analysis of the R. Baltica and B. marina genomes indicates the presence of putative genes encoding the three different enzyme subunits (PsrA, B, C) in both microorganisms. • Primers designed to target conserved sequences in the psrC gene of R. baltica and B. marina (Psr262F, Psr1162R) was used to test for the presence of a polysulfide reductase gene homolog in strain Zi62. • Phylogenetic analysis of the translated amino acid sequence of the PCR product obtained using Zi62 genomic DNA indicated that this peptide is most closely related to B. marina, and R. baltica PsrC subunits. • 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs (Pln46F/U1390R, and Eub338F-0-III/Pln930R suggested that an extremely diverse community of Planctomycetes is present at the spring. • Only a small fraction of the clones in this study (3 OTUs) were monophyletic with heterotrophic cultured representatives of this phylum (class Planctomyceteacea, genera Planctomyces, Pirellula,Blastopirellula, Rhodopirellula, Isosphaera, and Gemmata). • The majority of sequences described here using the Planctomycetes-biased primers were associated with candidate class WPS-1 (27 OTUs). These OTUs belonged to three different monophyletic lineages within this class (group a, b, and c in figure 1). Interestingly, many of the most similar clones to Zodletone OTUs have been reported to occur in anaerobic environments. • In addition, several non-Planctomycetes type rRNA clones were identified, and most of these phylotypes belonged to yet-uncultured bacterial candidate divisions that have previously been shown to be prevalent in anaerobic environments, such as candidate divisions OP11, OD1, WW1, WW2, WS3, OP3, in addition to Chlorobia, and Verrucomicrobia. putative psrC gene identified in Zi62 using PCR. The tree was constructed using partial (920bp) sequence of putative psrC amplified using primer pair 262F and 1162R (R. baltica) PsrC gene numbering). • 2. Acid production from sugars. • Strain Zi62 produced acids from carbohydrates (glucose, fructose, sucrose, trehalose, mannose, and xylose). • Succinate and acetate were the major products accumulating, with minor amounts of lactate, propionate, and formate. The pattern of product formation suggest a mixed acid fermentation pathway. • Repeated attempts to grow strain Zi62 under anaerobic fermentative conditions, either in complex (rumen fluid, yeast extract), or defined medium (glucose, sucrose, N-acetylglucosamine) were not successful. • Conclusions • An extremely diverse Planctomycetes population was identified in anaerobic, sulfide-saturated sediments at the source of Zodletone spring, suggesting that capability for anaerobic metabolism is widely distributed among members of this phylum. • A novel Planctomycete, strain Zi62 was isolated from the spring source using a complex soil-extract based medium. Strain Zi62 putatively represents a novel genus within the PRB clade within the Planctomycetaceae. • Strain Zi62 is capable of reducing elemental sulfur to sulfide, as well as acid production from sugars, suggesting sulfur reduction and carbohydrate fermentation as two possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions. References Staley, J. T. 1973. Budding bacteria of the Pasteuria-Blastobacter group. Can. J. Microbiol. 19:609-614. Schlesner, H. 1986. Pirella marina sp. nov., a budding peptidoglycan-less bacterium from brackish water. Syst. Appl. Microbiol. 8:177-1 Hirsch, P., and M. Muller. 1985. Planctomyces limnophilus sp. nov., a stalked and budding bacterium from fresh water. Syst. Appl. Microbiol. 6:276-2800. Fuerst, J. A., H. G. Gwilliam, M. Lindsay, A. Lichanska, C. Belcher, J. E. Vickers, and H. P. 1997. Isolation and molecular identification of planctomycete bacteria from postlarvae of giant tiger prawn, Penaeus monodon. Appl. Environ. Microbiol. 63:254-262. Giovannoni, S. J., J. E. Schabtach, and R. W. Castenholz. 1987. Isosphaera pallida, gen. nov., and comb. nov., a gliding, budding eubacterium from hot springs. Arch. Microbiol. 147:276-284 Wang, J., C. Jenkins, R. I. Webb, and J. A. Fuerst. 2002. Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater. Appl. Environ. Microbiol. 68:417-422 Chouari, R., D. L. Paslier, P. Daegelan, P. Ginestet, J. Weissenbach, and A. Sghir. 2003. Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant. Appl. Environ. Microbiol. 69:7354-7363. Dojka, M. A., P. Hugenholtz, S. K. Haack, and N. R. Pace. 1998. Microbial diversity in a hydrocarbon-and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64:3869-3877. Teske, A., K.-U. Hinrichs, V. Edgcomb, A. V. Gomez, D. Kysela, S. P. Sylva, S. M. L., and H. W. Jannasch. 2002. Microbial diversity of hydrothermal sediments in the Guaymas basin: evidence for anaerobic methanotrophic communities. Appl. Environ. Microbiol. 68:1994-2007. Elshahed, M. S., F. Z. Najar, B. A. Roe, A. Oren, T. A. Dewers, and L. R. Krumholz. 2004. Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide-and sulfur-rich spring. Appl. Environ. Microbiol. 70:2230-2239. Strain Zi62 represents a new genus within the Planctomycetes Strain Zi62 within the Pirellula – Rhodopirellula – Blastopirellula (PRB) clade within the family Planctomycetaceae. Strain Zi62 formed a monophyletic lineage with strong bootstrap support with a group of 10 Planctomycetes (98.8% to 96.7% sequence similarity). All 10 strains were isolated from marine habitats: (Postlarvae of the giant tiger prawn Penaeus monodon, tissue of the Aplysina sponge, Kiel Fjord, a marine aquarium, and Keauhou Beach, Hawaii) The closest recognized species to strain Zi62 is Blastopirellula marina (93.9% 16S rRNA gene sequence similarity). Strain Zi62 was 87.3% and 86.8% similar to the 16S rRNA genes of Pirellula staleyi and Rhodopirellula baltica the other two recognized species within the PRB clade Kiel fjord strain 116 (AJ231181) Aplysina sponge strain 16 (AF453519) Keauhou beach strain 611 (AJ231179) Marine aquarium strain 391 (AJ231180) Giant tiger prawn strain AGA M12 (X86391) Aplysina sponge strain18 (AF453518) Strain Zi62 Blastopirellula marina (X62912) Goal To determine the level of phylogenetic diversity as well as possible metabolic pathways utilized by members of the Planctomycetes in this anaerobic sulfide-saturated, hydrocarbon-impacted environment. Pirellula staleyi (AJ231183) Rhodopirellula baltica (BX294149) Planctomyces brasiliensis (AJ231190) Isosphaera pallida (AJ231195) Gemmata obscuriglobus (X56305) Planctomyces limnophilus (X62911) Candidatus “Kuenenia stuttgartien”(AF375995) Candidatus “Brocadia anammoxidans”(AF375994) Candidatus “Scalindua brodae”(AY254883) E. Coli (J01695)