R E S E A R C H A R T I C L E Free-living and aggregate-associated Planctomycetes in the Black Sea Clara A. Fuchsman1, James T. Staley2, Brian B. Oakley2, John B. Kirkpatrick1 & James W. Murray1 1School of Oceanography, University of Washington, Seattle, WA, USA; and 2Department of Microbiology, University of Washington, Seattle, WA, USA Correspondence: Clara A. Fuchsman, Abstract School of Oceanography, University of Washington, Seattle WA 98195-5351, USA. We examined the distribution of uncultured Planctomycetes phylotypes along Tel.: +206 543 9669; fax: +206 685 3351; depth profiles spanning the redox gradient of the Black Sea suboxic zone to e-mail: [email protected] gain insight into their respective ecological niches. Planctomycetes phylogeny correlated with depth and chemical profiles, implying similar metabolisms Present addresses: Brian B. Oakley, USDA within phylogenetic groups. A suboxic zone sample was split into > 30 and Agricultural Research Service, Athens, GA, < 30 lm fractions to examine putative aggregate-attached and free-living 30606, USA; Planctomycetes. All identified Planctomycetes were present in the > 30 m John B. Kirkpatrick, Graduate School of l Oceanography, University of Rhode Island, fraction except for members of the Scalindua genus, which were apparently Narragansett, RI, USA. free-living. Sequences from Candidatus Scalindua, known to carry out the anammox process, formed two distinct clusters with nonoverlapping depth Received 20 July 2011; revised 4 January ranges. One cluster, only 97.1% similar to the named species, was present at 2012; accepted 5 January 2012. high nitrite/nitrate and low ammonium concentrations in the upper suboxic zone. We propose this sequence type be named ‘Candidatus Scalindua richard- DOI: 10.1111/j.1574-6941.2012.01306.x sii’. A second cluster, containing sequences more similar to ‘Candidatus Scalin- dua sorokinii’, was present at high ammonium and low nitrite conditions in Editor: Alfons Stams the lower suboxic zone. Sequences obtained from the sulfidic zone (1000 m Keywords depth) yielded Planctomycetes from two uncharacterized Planctomycetacia clus- anammox; anoxic basin; particle-attached; ters and three potentially new genera as well as sequences from the uncultured uncultured Planctomycetes; OP3. OP3 phylum. anammoxosome, composed of tightly packed ladderane Introduction lipids, which is necessary to contain the volatile interme- Two pathways are involved in the conversion of fixed diate hydrazine (Sinninghe Damste et al., 2002). Internal nitrogen to N : the anammox process where NHþ cell compartments are a characteristic feature of the Plan- 2 4 þ NOÀ N via hydroxylamine and hydrazine intermedi- ctomycetes phylum (Lindsay et al., 2001), as are their pro- 2 ! 2 ates (van de Graaf et al., 1996, 1997) and denitrification tein envelopes and lack of a peptidoglycan cell wall 2 where 2NOÀ organic carbon=H S N CO =SO À (Liesack et al., 1986; Hieu et al., 2008). Owing to their 3 þ 2 ! 2þ 2 4 via a nitrous oxide intermediate (Zumft, 1997). Although internal compartments and compartmentalization a wide variety of bacteria can mediate denitrification (Zu- machinery, Planctomycetes have been proposed as the mft, 1997), only a subset of genera in the Planctomycetes progenitor of eukaryotes (Forterre & Gribaldo, 2010; OBIOLOGY ECOLOGY phylum are known to mediate the anammox process. In Reynaud & Devos, 2011). However, it seems more likely the marine environment, members of the Candidatus that similarities between Planctomycetes and eukaryotes Scalindua genus utilize the anammox process in both are a case of convergent evolution (Fuchsman & Rocap, sediments and oxygen-deficient water columns, and 2006; Strous et al., 2006; McInerney et al., 2011). MICR several other Candidatus genera (Kuenenia, Brocadia, Anammox bacteria are quite different from other char- Anammoxoglobus) mediate the process in freshwater and acterized Planctomycetes both phylogenetically and physi- wastewater treatment plants (Schmid et al., 2007). Anam- ologically. While anammox bacteria are primarily mox bacteria have an unusual internal compartment, the autotrophic, all of the other characterized Planctomycetes FEMS Microbiol Ecol && (2012) 1–15 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2 C.A. Fuchsman et al. are heterotrophic. Furthermore, the anammox bacterium summary, the Planctomycetacia consume organic matter Candidatus Kuenenia stuttgartiensis’s genome shares 0–30 under both aerobic and anaerobic conditions. Planctomycetes-specific genes with other Planctomycetes One bacterial strain has been isolated from a third genomes, as defined by reciprocal BLAST scores, compared class of Planctomycetes, the WPS-1 class, now known as to 275–764 between the other Planctomycetes genomes: Phycisphaerae. This recently cultured planctomycete, Rhodopirellula baltica, Blastopirellula marina, Gemmata Phycisphaera mikurensis, is a facultatively anaerobic obscuriglobus, and Planctomyces maris (Woebken et al., heterotroph and performs fermentation as well as nitrate 2007b). It should be noted, however, that these four reduction to nitrite (Fukunaga et al., 2009). Fermentation organisms are all in the same class, Planctomycetacia, may explain the ubiquity of Phycisphaerae at the sulfate- while the anammox genera form a separate class with, methane transition zone in marine sediments (Harrison and presumably share more genes with, the uncultured et al., 2009). members of the agg27 group (Elshahed et al., 2007). In aerobic environments, Planctomycetes have been Members of the Scalindua genus have a versatile found attached to sinking marine aggregates (DeLong metabolism. Two Scalindua species have been enriched et al., 1993), which is consistent with their role of degrad- from marine sediments and closely examined (van de ing complex organic matter. Stable isotope probing Vossenberg et al., 2008). Both of these Scalindua enrich- experiments indicate that members of Rhodopirellula ments and Candidatus Kuenenia stuttgartiensis, enriched metabolize phytodetritus (Gihring et al., 2009). Attach- from a wastewater treatment facility, have at least three ment to surfaces is important in the cell cycle of many metabolisms: the anammox process, iron and manganese Planctomycetes. In the cell cycle of Rhodopirellula, Blastop- reduction with formate, and nitrate reduction with irellula, Pirellula and Planctomyces, a bud on the mother formate (Strous et al., 2006; Kartal et al., 2007; van de cell turns into a motile swarmer cell with flagella, which Vossenberg et al., 2008). In the presence of nitrate, then differentiates into adult stalked nonmotile cells with Candidatus Kuenenia stuttgartiensis uses formate as an a holdfast, allowing cells to remain attached to a surface energy source for dissimilatory nitrate reduction to (Bauld & Staley, 1976; Schlesner et al., 2004; Gade et al., ammonium (DNRA); the ammonium is then converted 2005). For example, members of Pirellula have been to N2 using the anammox process (Kartal et al., 2007). found attached to marine diatoms during a diatom bloom Presumably, members of Scalindua use the same pathway. (Morris et al., 2006), P. mikurensis (WPS-1) was found Contrastingly, members of the class Planctomycetacia, attached to seaweed (Fukunaga et al., 2009), several Plan- including Rhodopirellula, Blastopirellula, Pirellula, and ctomycetes lineages were found attached to kelp (Bengts- Planctomyces, serve an important ecological function by son & Øvrea˚s, 2010), and members of the agg27 group performing the initial aerobic breakdown of complex were found on sinking marine particles (DeLong et al., organic matter into simpler compounds (Glockner et al., 1993). Owing to the presence of attached bacteria in all 2003). In culture, R. baltica and B. marina can degrade three classes of Planctomycetes, one might assume that sulfated heteropolysaccharides (Glockner et al., 2003; anammox bacteria in the marine environment are also Schlesner et al., 2004) as well as N-acetylglucosamine particle-attached. On the Namibia shelf, 24 ± 8% of (Rabus et al., 2002; Schlesner et al., 2004), a monomer of anammox cells visualized by CARD-FISH were particle- chitin and a subunit of bacterial peptidoglycan. Sulfated associated, while the majority of anammox cells were not heteropolysaccharides are abundant in the marine envi- (Woebken et al., 2007a). However, the size of these parti- ronment (Woebken et al., 2007b and references therein). cles were relatively small, and the particle maximum was Sulfatases, enzymes used to degrade sulfated heteropoly- close to the sediments, indicating that active anammox saccharides, are abundant in the genome of marine bacte- cells were likely resuspended with sediments (Woebken ria P. maris, R. baltica, B. marina (Glockner et al., 2003; et al., 2007a). Thus, it is yet to be determined whether or Woebken et al., 2007b), so this ability appears to be com- not anammox bacteria are associated with sinking parti- mon in the Planctomycetacia class. cles in open water low-oxygen systems. Some members of the Planctomycetacia class can also Planctomycetes living in suboxic and sulfidic water col- break down organic matter under both anaerobic and sul- umns have not been as well characterized. Here, we iden- fidic conditions. Blastopirellula strain Zi62 was isolated tify Planctomycetes of the Black Sea suboxic and sulfidic from a sulfur- and sulfide-rich spring and was found to waters. The Black Sea is a brackish semi-enclosed