Fuchsman C.A., J.T. Staley, B.B. Oakley, J.B. Kirkpatrick, J.W. Murray

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 perma- undergo fermentation and sulfur reduction (Elshahed nently stratified basin, with an oxic zone where the cold et al., 2007). The genes for fermentation exist in the intermediate layer (core density of r 14.5) represents h genomes of aerobic Planctomycetes R. baltica, B. marina, the lower boundary of direct communication with the and P. maris although these strains are not known to surface. Below this is a ~50-m-thick suboxic zone, where perform fermentation (Woebken et al., 2007b). In oxygen is < 10 lM and hydrogen sulfide is undetectable

(Murray et al., 1995). Below the suboxic zone is a sulfidic filtered onto a 0.2-lm Millipore Sterivex filters after pass- layer, which continues to the bottom. The diversity of ing through a 30-lm prefilter. Samples were frozen Planctomycetes in the Black Sea suboxic zone was previ- immediately and later stored at 22 °C. A set of DNA À ously examined using 16S rRNA gene sequencing (Kirk- samples were also prefiltered with 30-lm prefilters on the patrick et al., 2006). Bacteria were found related to the R/V Endeavor cruise in 2005. On the Bilim and Knorr Rhodopirellula, Planctomyces, and Candidatus Scalindua cruises, sampling focused in the suboxic region, but on genera as well as to several uncultured groups including the Endeavor cruise, a more complete depth profile of the agg27 and a cluster of sequences labeled ‘Unknown’ by oxic and sulfidic layers was obtained, including samples the authors (Kirkpatrick et al., 2006) and here labeled at 1000 m (rh = 17.19) and 2000 m (rh = 17.21). In Black Sea I. Using a different 16S rRNA gene primer set, addition, to look at the Planctomycetes community under Phycisphaerae (formerly WPS-1) have also been found in fully oxygenated conditions, 2 L from one Niskin bottle the Black Sea suboxic zone, and two members of the triggered at 50 m (rh = 13.9) was filtered onto a Sterivex WPS-1 group were categorized as aggregate-attached on the R/V AKBAHABT cruise 2900 in May 2007 in the (Fuchsman et al., 2011). Northeast Black Sea (44.5°N, 37.7°E), frozen immediately By examining depth profiles of Planctomycetes across and stored at 80 °C. À the redox gradient of the Black Sea suboxic zone, we aimed to gain insight into their metabolisms and ecologi- DNA extraction cal niches. With this goal, we examined Planctomycetes that are attached to sinking organic matter (> 30 lm The DNA extraction protocol was adapted from the study fraction) and those that are potentially free-living of Vetriani et al. (2003) and included 8–10 freeze–thaw (< 30 lm). Anammox bacteria are of particular interest cycles between a dry ice/ethanol bath and a 55 °C water for this study because of their known geochemical impor- bath, followed by chemical lysis with lysozyme and pro- tance in the Black Sea (Kuypers et al., 2003; Jensen et al., teinase K. DNA was then purified with phenol–chloro- 2008). form followed by spin columns (Qiagen, Valencia, CA).

Materials and methods 16S rRNA gene clones Owing to the salinity stratification of the Black Sea, char- Cloned 16S rRNA gene sequences were amplified from sul- acteristic inflections in the water-column profiles (such as fidic water at 1000 m depth from the western central gyre oxygen) are associated with specific density values regard- in March 2005. Some partial length sequences obtained less of when and where they were sampled (Murray et al., from suboxic water collected from the western central gyre 1995). Therefore, results presented here are plotted in April 2003 in the study of Kirkpatrick et al. (2006) were against potential density (rh) rather than depth (m). A further sequenced for this article [58F and 926R (Wang comparison of depth and density for each cruise is seen et al., 2002); complete insert equals 900 bp]: Genbank in Supporting information, Fig. S1. accession numbers: DQ368172, DQ368174, DQ368178, DQ368186, DQ368199, DQ368217–8, DQ368229, DQ36 8248, DQ368251, DQ368256, DQ368260, DQ368262, DQ3 Sampling 68269, DQ368274, DQ368283, DQ368292–3, DQ368295, DNA samples were collected in the western central gyre DQ368299, DQ368300, DQ368302, DQ368307–9, DQ3 of the Black Sea on four separate cruises: (1) in Septem- 68311, DQ368313, DQ368316, DQ368318, DQ368320, ber 2000 at station M10-L41 on the R/V Bilim (42°10′N, DQ368325, DQ368330–2. Sequencing was performed at 29°41′E), (2) in early June 2001 at station 2 Voyage 162 High-Throughput Sequencing Solutions (http://www. leg 17 of the R/V Knorr (42°30′N, 30°46′E), (3) in April htseq.org ) using primers T7 and M13R. Chromatograms 2003 at station 19 on Voyage 172 leg 7 of the R/V Knorr were hand inspected, and contigs assembled using the (42°30′N, 31°00′E), and (4) in late March 2005 at station Sequencher program (GeneCodes Corporation, Ann Arbor, 2 on cruise 403 of the R/V Endeavor (42°30′N, 30°45′E). MI). Resequenced suboxic zone sample sequences were For the Knorr and Endeavor cruises, samples were col- then processed in the REPK program (Collins & Rocap, lected using a CTD–Rosette with 10-L Niskin bottles and 2007) to choose which restriction enzymes should be used Sea Bird sensors. Approximately 2 L of seawater was fil- in terminal restriction fragment length polymorphism tered onto 0.2-lm Millipore Sterivex filters. Samples were (TRFLP) analyses, in order to ensure that groups of interest frozen immediately and later stored at 80 °C. For the could be distinguished with the restriction enzymes used. À R/V Bilim cruise, samples were obtained from 30-L 16S rRNA gene from 1000 m depth was amplified (58F and Nansen bottles, and approximately 7 L of water were 926R) and cloned as described in the study of Kirkpatrick

FEMS Microbiol Ecol && (2012) 1–15 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 4 C.A. Fuchsman et al. et al. (2006) using Invitrogen (Carlsbad, CA) TOPO vec- one unrelated bacterial species was reduced. In most of tors. Sequencing was first performed using 58F. Some the suboxic zone samples, there were 26–30 peaks in 1000-m clones were resequenced in full using primers T7 TRFLP chromatograms using MspI. Approximately 60% and M13R (fully sequenced clones identified by 1K: e.g. of peaks were identified in suboxic zone samples. In the 1K11E). All clones were chimera checked with Bellerophon lower suboxic zone (r 15.9), there were between 30 h (Huber et al., 2004) and submitted to GenBank (accession and 40 peaks, and the percentage of identified peaks numbers HQ883377-HQ883474). was lower. With HaeIII, there were also 25–40 peaks in TRFLP chromatograms, but HpyI881 only produced 20– 25 peaks. TRFLP The primers used in this study were designed to TRFLP chromatograms were obtained using Planctomy- amplify the Planctomycetes phylum. However, these prim- cetes-specific 16S rRNA gene primers (58F-FAM and ers have previously been found to also amplify sequences 926 R; Wang et al., 2002). Five samples from 2001 and affiliated with other members of the Planctomycetes– eight samples from 2003 were analyzed for TRFLP from Verrucomicrobia–Chlamydia superphylum [which include the suboxic zone of the western gyre as well as eight Lentisphaerae, candidate divisions Obsedian Pool 3 (OP3) prefiltered samples from 2000 and one whole sample and Poribacteria (Wagner & Horn, 2006)] as well as the from rh = 15.9. An additional sample from rh = 16.1 candidate division WS3 (Kirkpatrick et al., 2006). in 2001 could not be amplified well enough for TRFLP Although the database used to identify TRFLP peaks chromatograms to be obtained. In 2005, TRFLP chro- included clones representing Lentisphaera, WS3, Verruco- matograms were obtained across the redox gradient: microbia, and Chlamydia, previously found in clone one oxic sample, one hypoxic zone (39 lM) sample, libraries from the Black Sea, no actual peaks correspond- seven suboxic zone samples, and four sulfidic zone sam- ing to these phyla were identified. Identified TRFLP peaks ples. The chromatogram from MspI at rh = 16.4 was only corresponded to members of the Planctomycetes and rejected later in analysis because of insufficient total OP3 phyla. This difference between the communities peak height. Five suboxic zone samples that were prefil- obtained by sequencing and TRFLP could be due to steric tered with a 30-lm prefilter and one prefilter were also interactions of the fluorescent fluorescein amidite (FAM) examined. PCR products were amplified for 30 cycles at molecule attached to the forward primer used in TRFLP 60 °C, purified (QiaQuick columns; Qiagen, Valencia, or to the low abundance of the tangential phyla. CA) and digested separately overnight with restriction enzymes HaeIII, Hpy1881, MspI, and immediately pre- cipitated with ethanol. Fragment analysis was performed on a MegaBACE 1000 apparatus (Molecular Dynamics) at the University of Washington Marine Molecular Bio- Table 1. Bacterial 16S rRNA gene clones identified in TRFLP profiles technology Laboratory. Electrophoretic profiles were and their predicted fragment lengths for the enzymes used. Bold visualized with DAX software (Van Mierlo Software Con- indicates the peaks shown in depth profiles in Fig. 4 and Fig. S5 sultancy, the Netherlands). Phylotype Accession # Group HaeIII HpyI88I MspI

BO825 HQ883404 agg27 377 91 106 Identification of TRFLP peaks BO832 HQ883398 agg27 377 – 460 BO824 HQ883405 agg27 545 (543) – 460 The REPK program (Collins & Rocap, 2007) produced a JK616 DQ368233 Scalindua 236 530 260 list of predicted fragment lengths for 16S rRNA gene JK410 DQ368256 Scalindua 236 530 260 clones. These fragment lengths were ground truthed by JK215 DQ368316 Scalindua 236 530 260 two clones, corresponding to Planctomyces JK211 and JK742 DQ368186 Rhodopirellula 105 506 126 Candidatus Scalindua JK200, which were digested with BO815 HQ883400 Rhodopirellula 170 – 148 BO821 DQ368113 Planctomyces 174 83 131 all restriction enzymes individually. TRFLP peaks were JK221 DQ368320 Planctomyces 225 – 275 identified using the criteria that an identified peak was JK211 DQ368313 Planctomyces 281 412 115 ± 1 base pair from the length of the predicted fragment JK80 DQ368293 Black Sea I 280 270 308 length for all restriction enzymes [in two cases, the JK589 DQ368217 Black Sea I 471 (473) – 150 identified peak was 2 base pairs from the predicted frag- JK782 DQ368199 Black Sea I 582 291 121 ment (shown in parentheses in Table 1)]. An identified BO813 HQ883399 Black Sea I – 288 326 peak was allowed a total range of 1 base pair BO823 HQ883406 Black Sea I – 288 211 1K2C HQ883379 OP3 232 390 – between samples. Using a small bin size, the risk that an JK79 DQ368292 OP3 266 555 – identified TRFLP peak actually represented more than

tion by atmospheric oxygen. During Bilim 2000 and Statistical analyses Knorr 2001, nutrients were analyzed on shipboard by TRFLP profiles were normalized downward by total peak S. Tugrul (METU, Turkey). For Knorr 2003 and Endeavor height with a minimum cutoff of 0.3% of the total peak 2005, nutrients were analyzed shipboard using a Techni- height. TRFLP peaks were binned using frame shifting con Autoanalyzer II system. Nitrate was reduced to (Hewson and Fuhrman, 2006) with four frames at 0.5 nitrite using a cadmium column, which was measured base pair intervals. For each enzyme, resemblance matri- using sulphanilamide and N(1-naphthyl)-ethylenediamine ces were obtained using the Whittaker index, which takes (Armstrong et al., 1967). Ammonium was analyzed using abundance (peak height) into account, and the Jaccard the indophenol-blue procedure (Slawky & MacIsaac, index, which uses presence/absence. Trends were the same 1972). Deep-water ammonium samples were diluted with for both indices, so only the Whittaker index, which con- nutrient-free Black Sea surface water to reduce sulfide tains more information, is discussed. For each compari- content. son between two samples, the maximum similarity of the four frames was used. Hierarchical cluster analyses (using Results the group average) and principal component analysis (PCA) were performed using the Primer 6 program. The Chemistry restriction enzyme MspI was primarily used in these analyses because it distinguished the greatest number of phyl- Oxygen and sulfide data for each cruise are discussed in otypes in the suboxic zone samples. Data from the HaeIII the study of Fuchsman et al. (2008). The boundaries of enzyme formed the same large clusters but with slightly the suboxic zone for each year, < 10 lM O2 for the higher percentage of similarities. Owing to prefiltration of upper boundary and first appearance of detectable sulfide most samples from 2000, they could not be included in for the lower boundary, are indicated by dashed lines in these analyses. Error in the resemblance matrix and sig- Fig. 1. These density surfaces were slightly shoaled in nificance level of the cluster diagram were determined 2003 compared to 2000, 2001, and 2005 (Fig. S1). using a Monte–Carlo simulation of 50 replicates using Nitrite concentrations were low through the oxycline the average error of both normalized peak height and peaked in the lower suboxic zone (Fig. 1). In 2000

(± 83 rfu where total peak height is 15 000 rfu) and base and 2005, the nitrite maximum was at rh = 15.85, while pairs (± 0.06 bp) as determined by 14 sets of duplicate in 2003, it was at rh = 15.8, and it was absent in 2001. TRFLP profiles. All simulated replicates had at least 85% Nitrite concentrations from 2000 were significantly larger Whittaker similarity. than in other years, with a maximum at 0.28 lM. The maxima were < 0.1 lM in the other years (Fig. 1). While the flux of ammonium across the sulfide bound- Phylogenetic trees ary into the suboxic zone was similar between years Trees were constructed in MEGA4 (Tamura et al., 2007) (Fuchsman et al., 2008), measurable ammonium reached after aligning with CLUSTALX (Larkin et al., 2007). In the different depths in the suboxic zone (Fig. 1). In 2000, phylogenetic analyses where multiple sequence lengths ammonium became undetectable at rh = 16.05, in 2001 were necessary, trees were constructed using the mini- at rh = 15.95, in 2003 at rh = 15.8, and in 2005 at mum evolution method (Rzhetsky & Nei, 1992) with rh = 15.85. 1000 bootstraps, and distances were computed using the maximum composite likelihood method (Tamura et al., Molecular biology 2004). These multiple sequence alignments contained complete sequences and were not trimmed down to par- Bacteria identified in TRFLP chromatograms and their tial Black Sea sequences to maintain previously published respective fragment lengths are shown in Table 1. The phylogenetic relationships. The neighbor-joining tree in phylogenetic relationships of these identified Planctomyce- Fig. 5 was created using 720-bp sequences with 100 boot- tes are shown in Fig. 2. All the Planctomycetes identified straps. in the suboxic zone in 2005 were found on particulate matter (> 30 lm) except for Scalindua, which were only found in the prefiltered sample (< 30 m; Fig. 3). Nutrient concentrations l A hierarchical cluster analysis of the similarity in S. Konovalov and A. Romanov (MHI, Ukraine) mea- TRFLP samples from the western central gyre suboxic sured oxygen with the Winkler method and assayed sul- zone were compared using Whittaker indices, based on fide by iodometric titration (Cline, 1969). In both cases, restriction digests with enzyme MspI. The Planctomycetes/ reagents were bubbled with argon to avoid contamina- OP3 community separated into oxic (rh = 14.9–15.3),

(a) NO - (µM) (b) - 2 NO2 (µM) 0.05 0.1 0.15 0.2 0.25 0.3 0 0.04 0.08 0.12 0.16 0.2

N2 Excess (µmol/kg) N2 Excess (µmol/kg) 8 12 16 20 4 6 8 10 12

15.4 15.4 15.6 15.6

a 15.8 t 15.8 e h

T 16 16 a m

g 16.2 16.2 i S 16.4 TRFLP 16.4

N2 Excess 16.6 - 16.6 NO2 2001 2000 NH + 16.8 4 16.8 0 2000 4000 6000 0 1000 2000 3000 Normalized TRFLP Peak Height Normalized TRFLP Peak Height 0 4 8 12 16 20 0 4 8 12 16 20 + + NH4 (µM) NH4 (µM)

- - (c) NO2 (µM) (d) NO2 (µM) 0 0.04 0.08 0.12 0.16 0.2 0 0.04 0.08 0.12 0.16 0.2

N2 Excess (µmol/kg) N2 Excess (µmol/kg) 4 6 8 10 12 4 6 8 10 12

15.4 15.4 15.6 15.6 a

t 15.8 15.8 e h

T 16 16 a m

g 16.2 16.2 i S 16.4 16.4 16.6 16.6 2003 2005 16.8 16.8 0 1000 2000 3000 0 1000 2000 3000 Normalized TRFLP Peak Height Normalized TRFLP Peak Height 0 4 8 12 16 20 0 4 8 12 16 20 + + NH4 (µM) NH4 (µM)

Fig. 1. Depth proﬁles of TRFLP peak height for Candidatus Scalindua (MspI) compared to reactants (NO2À and NH4þ) and product (biological N2; Fuchsman et al., 2008) of the anammox process: (a) in 2000 (b) 2001 (c) 2003 (d) 2005. Note different scale for axes for 2000. Average error for peak height is ± 83 rfu. Dashed lines denote suboxic zone boundaries. Nutrients and DNA sequences were collected from the same cast where possible.

upper suboxic (rh = 15.6–15.85), lower suboxic (rh = MspI or HaeIII were examined (Fig. S2). Each set of 15.9–16.0), and sulfidic [rh = 16.1–17.21 (2000 m)] clus- samples clustered most closely with the other samples ters (Fig. S2). Although no sulfide was detected at from the same cruise, excepting samples in the rh = rh = 16.1 2005, the rh = 16.1 2005 sample clustered with 15.75–15.85 range from 2003 to 2005, which clustered the sulfidic samples when either restriction enzymes MspI together. or HaeIII were examined. However, sulfide was detected Hierarchical clusters indicated variability in the suboxic 3 m below this sample, and the detection limit for sulfide zone Planctomycetes community. A PCA was used to find with this technique is 3 lM (Konovalov et al., 2003), so which TRFLP peaks contributed the greatest microbial sulfide may have been present at rh = 16.1 at low variability between depths and cruises (Fig. S3). For concentrations. Although suboxic zone samples from MspI, eigenvector PC1, representing 37% of the variabil- 3 years were analyzed, they all separated into lower and ity, was strongly correlated with peak 210 (BO823 BSI; upper suboxic zone clusters at rh = 15.9 when either linear coefficient of 0.783), which was particularly high in

Fig. 2. Minimum evolution phylogenetic tree representing the Planctomycetes. Bold orange sequences are from the sulfidic zone (1000 m). Phylotypes identified in TRFLP profiles are bold and color-coded by their aggregate affinity: free-living (red), aggregate-attached (green), equal in both fractions (blue) or unable to determine (black). Bootstrap support values > 50% are shown. suboxic zone samples from 2001 (Fig. 4), and also correlated ples from 2003 to 2005 and low in 2001 (Fig. 4), while with peak 259 (Scalindua; linear coefficient 0.3), which peak 127 was absent in 2003 samples and abundant in was abundant for lower suboxic zone samples. Eigenvec- 2001 samples. BO832, Scalindua, and peak 370 (which tor PC2, representing 16% of the variability, indicated matches peak 127) also contributed the most to microbial opposite trends for peak 459 (BO832 agg27) and peak variability when peaks cut with the HaeIII enzyme were 127 (linear coefficients of 0.554 and 0.584, respectively); examined (data not shown). BO823 is not cut by the À BO832 agg27 was abundant in upper suboxic zone sam- HaeIII enzyme.

spond to OP3 groups II, III, and IV as defined by Glock- ner et al. (2010). The most abundant sequence group (phylotypes 1K2C from OP3-III) was also identified in TRFLP profiles from the sulfidic zone with its highest normalized peak height at 1000 and 2000 m (Figs S4 and S5). OP3 phylotype JK79, also in OP3-III, had a contrast- ing depth profile where peak height was highest at

rh = 16.4 in the upper sulfidic zone (Fig. S5). The Planctomycetes sequences from 1000 m in the sulfidic zone clustered into group A17 and in uncharacterized sulfidic group A in the class Planctomycetacia and in a series of small clusters in the deeply branching class of Planctomycetes (Fig. 2). Interestingly, sequences 1K8B and 1K11E from 1000 m, in the A17 group, are closely related to sequences found in a diatom bloom in oxic ocean surface waters (Morris et al., 2006) and from the suboxic zone of Lake Tanganyika (Schubert et al., 2006), implying a wide range of ecological niches in the A17 group. How- ever, Planctomycetes from 1000 m primarily are found in Fig. 3. Comparison of Planctomycetes from particulate and filtrate uncharacterized groups outside the Planctomycetacia class. samples. Percentage of normalized TRFLP peak height found on a These Planctomycetes imply that novel genera and perhaps

30 lm prefilter from rh = 15.8 (black) compared to in the filtrate novel metabolic groups of Planctomycetes reside in sulfidic (grey). Bars are labeled by their corresponding fragment length (MspI) environments. and if identified, by their name and taxonomy.

Discussion Oxic zone Particle-attached and free-living bacteria Some Planctomycetes are found both in oxic and suboxic zones. Comparing the various suboxic zone samples to a As aggregates larger than 53 lm dominate the vertical sample with 105 lM oxygen (rh = 14.9), seven phylo- mass flux in the ocean (Clegg & Whitfield, 1990; Amiel types were found in common: agg27 BO832, BSI BO813, et al., 2002), bacteria caught on the 30-lm-pore-size filter BSI BO823, BSI JK589, Planctomyces JK211, Planctomyces collected from the center of the suboxic zone in 2005

BO821, Planctomyces JK221, Rhodopirellula JK742. Three (rh = 15.8) were likely mainly attached to sinking aggre- of these phylotypes (BSI BO813, Planctomyces JK211, gates or large suspended particles. These particles con- Planctomyces BO821) were also identified in a fully oxy- tained diatom chloroplasts (Fuchsman et al., 2011). genated sample (294 lM O2 and 0.6 lM NO3À; E. Yaku- Particulate organic carbon (determined by a 0.7-lm- shev, P.P. Shirshov Institute of Oceanology, Russia, pore-size filter) was around 4 lM in the suboxic zone at unpublished data) from 50 m depth (rh = 13.9) in the that time, and C/N ratios of these particles were about 9, Northeast Black Sea in May 2007. These two oxygenated indicating that the particulate organic matter was at least Planctomycetes communities shared only 22% similarity partially degraded (Fuchsman et al., 2011). Particulate using the Whittaker Index, but as they also differed in manganese had a maximum at rh = 15.85 and was location, time, and depth, we cannot pinpoint the cause detectable throughout the suboxic zone (Fuchsman et al., of these community differences. 2011). Identified 16S rRNA gene phylotypes were categorized as particle-attached or free-living based on a comparison Sulfidic zone of TRFLP chromatograms between the prefiltered sample

Planctomycetes-specific 16S rRNA gene primers amplified and the 30-lm-pore-size prefilter from rh = 15.8 2005 Planctomycetes, Lentisphaera, and OP3 clones from (Fig. 3). If > 75% of the combined normalized peak 1000 m (Fig. 2 and Fig. S4). These primers are known to height for a phylotype was found on the prefilter chro- amplify some sequences from phyla in the Planctomycetes matogram, it is considered to be particle-attached. If –Verrucomicrobia–Chlamydia superphylum (Kirkpatrick > 75% of the combined peak height for a phylotype was et al., 2006). A large number of OP3 sequences were found in the filtrate chromatogram, it is considered free- obtained from 1000 m (Fig. S4). These sequences corre- living. These findings were supported and extended by

14.8 Planctomyces Planctomyces Planctomyces JK211 JK221 BO821 15.2

15.6 theta

16 Sigma 16.4 2001 2003 2005 16.8 0 1000 2000 3000 4000 5000 0 400 800 1200 1600 2000 0 400 800 1200 1600 2000 14.8 Agg27 Agg27 Agg27 BO832 BO824 BO825 15.2

15.6 theta

16 Sigma

16.4 2001 2003 2005 16.8 0 400 800 1200 1600 2000 0 400 800 1200 1600 2000 0 400 800 1200 1600 2000 14.8 Rhodopirellula BO815 Black Sea I JK589 15.2 Rhodopirellula JK742

15.6

theta 16

16.4 Sigma

16.8 2001 2003 17.2 2005 0 1000 2000 3000 40000 400 800 1200 1600 2000 0 400 800 1200 1600 2000 14.8 Black Sea I Black Sea I JK782 BSI Black Sea I BO823 JK80 15.2 BO813

15.6 theta

16 Sigma

16.4 2001 2003 2005 16.8 0 400 800 1200 1600 2000 0 400 0 2000 4000 0 2000 4000 6000 Normalized TRFLP peak height Normalized TRFLP peak height

Fig. 4. Depth proﬁles of TRFLP peak height from 2001, 2003 and 2005, with restriction enzymes as per Table 1. Names are color-coded by aggregate afﬁnity: aggregate-attached (green), free-living (red), found in both fractions (blue) or not determined (black), and presence in fully oxygenated sample (box). Average peak height error is ± 83 rfu.

66 Black Sea JK582 (DQ368210) Black Sea JK634 (DQ368246) Black Sea JK529 (DQ368208) Black Sea JK615 (DQ368232) Black Sea JK598 (DQ368224)

Black Sea JK524 (DQ368205) Candidatus Black Sea JK584 (DQ368212) Black Sea JK587 (DQ368215) Black Sea JK594 (DQ368220)

Black Sea JK610 (DQ368228) scalindua richardsii Black Sea JK611 (DQ368229) 58 Black Sea JK633 (DQ368245) Black Sea JK626 (DQ368241) Black Sea JK639 (DQ368250) 63 Black Sea JK614 (DQ368231) Black Sea JK585 (DQ368213)

Black Sea JK590 (DQ368218) Black Sea JK588 (DQ368216) 51 Black Sea JK528 (DQ368207) Black Sea JK625 (DQ368240) 100 Black Sea JK616 (DQ368233) Black Sea JK632 (DQ368244) Black Sea JK630 (DQ368243) Black Sea JK597 (DQ368223) 100 Black Sea JK586 (DQ368214) Black Sea JK622 (DQ368239) 100 60 Black Sea JK609 (DQ368227) Black Sea JK583 (DQ368211) Black Sea JK636 (DQ368247) 87 Black Sea BlackSea_3 (EU478626) Black Sea JK84 (DQ368297) Peru OMZ cluster III Peru_41 (EU478669) Peru OMZ cluster I Peru_43 (EU478688) 100 Black Sea JK456 (DQ368270) 53 Black Sea JK198 (DQ368306) Anammox 15.6 15.8 Black Sea JK200 (DQ368308) 87 group II 15.9/16.0 Black Sea JK410 (DQ368256)

Namibia OMZ cluster Namibia_174 (EU478643) 50 Peru OMZ cluster II Peru_27 (EU478686) Black Sea Candidatus Scalindua sorokinii (AY257181) 99 Black Sea JK215 (DQ368316) 59 Cand. S. sorokinii Black Sea JK461 (DQ368272) 59 0.002 Black Sea JK854 (DQ368148)

Fig. 5. A bootstrapped neighbor-joining phylogenetic tree of Black Sea anammox 720 bp sequences which shows a new Candidatus species, ‘Candidatus Scalindua richardsii.’ Sequences where 869 bp are available are underlined. Density level is noted by symbol. comparing presence and absence in prefiltered and bulk bian shelf, 24 ± 8% of anammox cells were associated water samples rh = 15.7–15.9. Some Planctomycetes were with small particles, likely resuspended sediment (Woeb- identified as particle-attached including Planctomyces ken et al., 2007a). With our dataset, we cannot eliminate phylotype JK221; agg27 phylotypes BO825 and BO832; the possibility that anammox bacteria exclusively attach and BSI phylotypes BO823, JK782, JK80, JK589, and to < 30-lm particulate material. However, they certainly BO813. Other Planctomycetes were found equally in both do not attach to sinking particles (defined as > 53 lm by fractions, including Planctomyces phylotype BO821, Rho- Clegg & Whitfield, 1990). dopirellula BO815, the unidentified peak pair 370/127 Some Planctomycetes were found in a range of oxygen (HaeIII/MspI), and unidentified peak 166 (MspI). These concentrations from fully oxygenated to suboxic waters organisms may be attached to both large and small (such as Planctomyces JK211, BO821 and BSI BO813), (< 30 lm) particles or live some of their life cycle as while others were found at 105 lM oxygen and suboxic free-living organisms (Bauld & Staley, 1976; Schlesner waters (such as agg27 BO832 and BO825 and Planctomy- et al., 2004; Gade et al., 2005). In contrast, TRFLP peak ces JK221). As all these Planctomycetes were found on par- 259 corresponding to the Scalindua phylotype was the ticles, they may have been attached to sinking particles in only peak that was only present in the filtrate, and thus oxic waters and remained on the particles as they sank Scalindua are classified here as free-living. Planctomyces into the suboxic zone. Other Planctomycetes were present JK211, although present at low levels on the particulate only under suboxic conditions (BSI JK782, JK80, BO823, material, was also classified as free-living. On the Nami- and Rhodopirellula BO815; Fig. 4), implying a community

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2012) 1–15 Published by Blackwell Publishing Ltd. All rights reserved Planctomycetes in the Black Sea 11 specific to particles in low-oxygen waters. These Plancto- profiles of BSI group phylotypes had maxima in the cen- mycetes either have a rapid chemotactic response to nutri- ter of the suboxic zone but were not present in the sulfi- ent plumes generated by sinking particles as seen in some dic zone (Fig. 4) and were generally not found under other marine bacteria (Stocker et al., 2008), or they create higher-oxygen conditions. The similarities between BSI their own particles from processes such as manganese TRFLP depth profiles and nitrate and particulate manga- oxidation. Manganese- and iron-encrusted Planctomycetes nese imply involvement in nitrate or metal cycling. The have been found in lakes, although these organisms have depth range of the BSI phylotype’s TRFLP peak height not yet been well characterized using molecular methods coincides with the presence of particulate manganese oxi- (Schmidt et al., 1981, 1982). des; the maximum in particulate manganese was at

rh = 16.05 in 2001 (Konovalov et al., 2003), rh = 15.8 in 2003 (Trouwborst et al., 2006), and r = 15.85 in 2005 Metabolic implications h (Fuchsman et al., 2011). Genomic and cultivation studies in the open ocean imply that most Planctomycetes, excluding anammox bacteria, Anammox are oxygenic heterotrophs able to degrade complex organic compounds (Glockner et al., 2003; Schlesner Candidatus Scalindua sorokinii has been implicated in the et al., 2004; Woebken et al., 2007b). Rhodopirellula-like anammox process in the suboxic zone of the Black Sea JK742, which is only detectable in samples from 2005 (Kuypers et al., 2003). The substrates for the anammox with more than 5 lM oxygen (Fig. 4), may be such an process are readily available in the Black Sea. Nitrite is organism. However, the suboxic and sulfidic zones of the produced by nitrate reduction linked to organic matter Black Sea are an ideal place to find Planctomycetes with degradation (Ward & Kilpatrick, 1991). There is a con- alternative metabolisms. For example, the Rhodopirellula- stant flux of ammonium from the sulfidic layer into the like phylotype BO815 was not found in oxic samples but suboxic zone. This ammonium can be autotrophically had a maximum in the rh = 15.7–15.95 region and was oxidized to nitrite even at extremely low oxygen concen- detectable throughout the sulfidic zone (Fig. 4). This trations (Ward & Kilpatrick, 1991; Lam et al., 2007). organism, as well as the other Planctomycetacia sequenced Ammonium oxidation to nitrite can then be linked to the from 1000 m (> 300 lM H2S; Fig. 2), may be carrying anammox process (Lam et al., 2007) allowing anammox out fermentation in the sulfidic zone, as noted previously to proceed under low-organic-matter conditions (Fuchs- in a sulfur spring by isolate Zi62 (Elshahed et al., 2007). man et al., 2008). The reactants and products of the Organic acids, indicative of fermentation, have a maxi- anammox process, along with the Scalindua normalized mum in the Black Sea’s upper sulfidic zone and are pres- peak height, are seen in Fig. 1. We used normalized ent throughout the lower sulfidic zone (Albert et al., TRFLP peak height as a proxy for the relative abundance 1995). of Scalindua. Owing to the prefiltering of DNA samples We used depth profiles across the redox gradient to from 2000, peak heights from this year cannot be com- investigate linkages to substrates available to these organ- pared to other years. Biological N2 (N2 excess) was calcu- isms. Consistent vertical profiles throughout the 3 years lated by subtracting the N2 present because of physical examined indicate robust trends in the distribution of processes such as gas solubility and mixing in the study Planctomycetes in the suboxic zone (Fig. 4). The genera of Fuchsman et al. (2008). Scalindua peak height and

Planctomyces, agg27, and Black Sea I (BSI) had fairly con- concentration of biologically produced N2 generally corre- sistent depth profiles between identified genotypes within late (R2 = 0.6). the group (Fig. 4). Agg27 clones were generally present In 2000 and 2003, two maxima were seen in the depth in the upper suboxic zone and the oxic zone (Fig. 4). profile of Scalindua peak height: one at the nitrite maxi- A microaerophilic enrichment culture of an agg27 strain mum and the second at the bottom of the suboxic zone. sampled in 2003 grew in Black Sea water enriched with In 2005, no DNA samples were collected at the nitrite ammonium and nitrite (Kirkpatrick et al., 2006). maximum. These two TRFLP peak maxima may corre- Although organic matter was present in the Black Sea spond to different ecotypes of Scalindua. All the Scalin- water, no additional organic compounds were added. The dua sequences obtained from the Black Sea have the same enrichment in this medium implies, but does not guaran- TRFLP cut pattern with the restriction enzymes used in tee, the use of nitrite or ammonium in its metabolism. this study. However, Scalindua clones sequenced from the The ability to utilize oxygen at a large range of concentra- 2003 cruise (Kirkpatrick et al., 2006; 720 bp) form three tions or to switch to nitrate reduction under low-oxygen clusters (Fig. 5). For five select longer sequences, similar- conditions may explain the existence of Planctomyces in ity was obtained using MatGat 2.0 over 869 base pairs. both oxic and suboxic zones. Contrastingly, the depth These longer sequences are used to represent each cluster.

JK215 was > 99% similar to Candidatus S. sorokinii, a anammox activity in the upper suboxic zone of the Black named sequence originally recovered from the lower sub- Sea (Jensen et al., 2008), some anammox bacteria can toler- oxic zone of the Black Sea (Kuypers et al., 2003), and ate sulfide (Kalyuzhnyi et al., 2006). Our data suggest that they are in the same cluster (Fig. 5). Another cluster of different species of Scalindua may be found in the upper sequences represented by phylotype JK410 was 2.2% dif- and lower suboxic zone, leaving the possibility of sulfide ferent from Candidatus S. sorokinii. This group was only tolerant Scalindua mediating the anammox process in the found in the lower suboxic zone (rh = 15.9 and 16.0) lower suboxic zone. Scalindua may alternatively catalyze under conditions of high ammonium concentration and metal reduction (van de Vossenberg et al., 2008) in the low nitrite concentration. However, bootstrap analysis of upper sulfidic zone. At rh = 16.2 in 2000, particulate man- the neighbor-joining phylogenetic tree could not deter- ganese was < 1 nM but particulate iron was 6 nM (Yigiter- mine that the Candidatus S. sorokinii cluster and the han et al., 2011). The Black Sea Sulfurimonas, a bacterium JK410 cluster were significantly different (Fig. 5). A third closely related to autotrophic denitrifiers and found to be cluster, represented by 869-bp sequence JK616, was 2.9% active in the upper sulfidic zone (Glaubitz et al., 2010), different from Candidatus S. sorokinii and 1.8% different may also be responsible for N2 production below the sub- from JK410. The mean difference between Candidatus S. oxic zone. Sulfurimonas DNA was found from rh = 16– sorokinii and all the shorter sequences in the JK616 clus- 16.2 in 2000 using TRFLP with universal bacterial primer ter was 2.5%. Sequences in the JK616 cluster were only 27F (unpublished data, CAF). Thus, Scalindua and Sulfuri- found at rh = 15.8, the nitrite maximum where nitrate monas coexisted in this depth range. Future work should was in high concentration and ammonium was in very examine more closely N2 production pathways at the tran- low concentration. This group was statistically different sition between suboxic and sulfidic zones. from the JK410 and Candidatus S. sorokinii clusters (Fig. 5) as well as from Peru clusters I, II, and III and the Conclusions Namibian cluster from the study of Woebken et al. (2008). Candidatus Scalindua arabica (Woebken et al., In this study, we examined the spatial and temporal dis- 2008) was 5% different from this cluster. tribution of the many uncultured Planctomycetes in the As JK616 is sufficiently divergent from the named spe- Black Sea. The Planctomycetes in the same phylogenetic cies ‘Candidatus S. sorokinii’ to be named a new species groups generally had similar depth profiles, implying sim- (Stackebrandt & Goebel, 1994), we are proposing a new ilar metabolisms within phylogenetic groups. However, species ‘Candidatus Scalindua richardsii’, named in honor these potential metabolisms were clearly more diverse of Francis A. Richards, a chemical oceanographer who than those found in cultured Planctomycetes. hypothesized the existence of anaerobic ammonium oxi- All cultured Planctomycetes have been found to attach dation based on chemical fluxes (Richards, 1965). The to surfaces. Organic aggregates are sources of abundant depth distribution of ‘Candidatus S. richardsii’ suggests but transitory nutrients. Theoretically, heterotrophic that it is adapted to extremely low ammonium concentra- aggregate-attached bacteria should be adapted to consume tions. It is possible that it can perform DNRA, an ability substrates quickly and reproduce rapidly while adaptation found in other anammox bacteria (Kartal et al., 2007; van to life at free-living energy-limited conditions includes de Vossenberg et al., 2008). In contrast, the JK410/Can- efficient use of resources and high affinity transporters for didatus S. sorokinii cluster appears to be adapted to acquiring nutrients. All identified Planctomycetes in this extremely low nitrite concentrations, but where ammo- study were present on aggregates except for members of nium is available. They may be the anammox bacteria Scalindua known to facilitate the anammox reaction. Scal- that were found to be closely linked to active gammapro- indua were only found in the < 30 lm fraction and teobacterial ammonium oxidizers in the lower suboxic existed in conditions that require the ability to acquire zone (Lam et al., 2007). reactants at nanomolar concentrations: ‘Candidatus S.

In all four cruises, biological N2 profiles imply small richardsii’ (phylotype JK616) was present at high nitrite amounts of N2 production may occur in the presence of and nitrate and low ammonium concentrations, while a low concentrations of sulfide (rh = 16.1–16.25; Fig. 1). ‘Candidatus S. sorokinii’ sequence cluster was present at Scalindua DNA was found in the upper sulfidic layer high ammonium and low nitrite concentrations. The fact

(9.5 lM sulfide at rh = 16.2 in 2000). At depths below this that Scalindua bacteria in the Black Sea are free-living (20 lM H2S at rh = 16.4), Scalindua peak heights were and not linked to sinking organic matter is consistent undetectable. This finding is consistent with the presence of with their activity under oligotrophic conditions (Fuchs- ladderane lipids associated with anammox bacteria at man et al., 2008). This situation contrasts to that of het- depths with detectable sulfide in 2003 (Wakeham et al., erotrophic denitrification, which is stimulated by high 2007). While 1–2 lM sulfide has been shown to inhibit organic matter conditions (Ward et al., 2008).

Acknowledgements Forterre P & Gribaldo S (2010) Bacteria with a eukaryotic touch: a glimpse of ancient evolution? P Natl Acad Sci USA We would like to thank B. Paul for collecting DNA sam- 107: 12739–12740. ples from 2000 to 2001 and for nutrients data from 2005 Fuchsman CA & Rocap G (2006) Whole-genome reciprocal and S. Konovalov (MHI, Ukraine) and S. Tugrul (METU, BLAST analysis reveals that planctomycetes do not share an Turkey) for use of oxygen and nutrient data. We would unusually large number of genes with Eukarya and Archaea. like to thank an anonymous reviewer and G. Rocap for Appl Environ Microbiol 72: 6841–6844. helpful comments on the manuscript. C.A.F. and J.B.K. Fuchsman CA, Konovalov SK & Murray JW (2008) were supported by IGERT traineeships in Astrobiology Concentration and natural stable isotope profiles of nitrogen under NSF grant 05-04219. This work was also supported species in the Black Sea. Mar Chem 111: 90–105. by NSF OISE 0637866, OISE 0637845, and OCE 0649223. Fuchsman CA, Kirkpatrick JB, Brazelton WJ, Murray JW & Sampling in May 2007 was supported by a Lewis and Staley JT (2011) Metabolic strategies of free-living and Clark Astrobiology Travel Grant and was accomplished aggregate associated bacterial communities inferred from biological and chemical profiles in the Black Sea suboxic with the help of Evgeniy Yakushev (CRDF Grant # CGP- zone. FEMS Microbiol Ecol 78: 586–603. 15123). The authors have no conflict of interest to Fukunaga Y, Kurahashi M, Sakiyama Y, Ohuchi M, Yokota A declare. & Harayama S (2009) Phycisphaera mikurensis gen. nov., sp. nov., isolated from a marine alga, and proposal of References Phycisphaeraceae fam. Nov., Phycisphaerales ord. Nov. and Phycisphaerae classes nov. in the phylum Planctomycetes. 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