High Phylogenetic Diversity in a Marine-Snow- - Associated Bacterial Assemblaae

AQUATIC MICROBIAL ECOLOGY Vol. 14: 261-269,1998 Published April 6 Aquat Microb Ecol

High phylogenetic diversity in a marine-snow- - associated bacterial assemblaae

Johannes Rathl,*, Ke Ying wu2, Gerhard J. ~erndl~,Edward F. DeLong4

'Molecular Biology Laboratory, Institute of Zoology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria 'Department of Ecology. Evolution and Marine Biology, University of California. Santa Barbara, California 95616, USA 3Department of Biological Oceanography, Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands 4Monterey Bay Aquarium Research Institute, PO Box 628, 7700 Sandholdt Rd. Moss Landing, California, USA

ABSTRACT: Large p500 pm), suspended particles of organic and inorganic material, often referred to as 'marine snow', are considered important in the vertical transport and biogeochemical transformation of particulate organic carbon in the marine environment. Previous work has indicated that bacterial species inhabiting marine snow particles may differ greatly from those commonly found living free in the surrounding water column. To further characterize marine-snow-attached bacterial populations, we sampled marine snow by SCUBA diving during periods of intense macroaggregate formation in the northern Adriatic Sea in August 1991. Small subunit ribosomal DNA (rDNA) fragments of bacteria were amplified from extracted nucleic acids by the polymerase chain reaction (PCR). The diversity of the recovered rDNA clones was initially assessed by cornpmg restriction fragment length polymorphisms (RFLPs) of individual clones. Ninety-five bacterial clones examined yielded 90 different KFLP patterns, representing an estimated sampling coverage of only 5.3%. Sequence analysis of 39 randomly chosen clones was used to assess the general phylogenetic affiliation of individual clones. Bacterial phyla represented in the library included affiliates of the Planctomyces, the Gram-positive bacteria, the Cytophaga-Flavobacteria-Bacteroides (CFB) lineage, and the alpha-, gamma-, delta-, and epsilon-subdivisions of the Proteobacteria. The results suggest that bacterial colonization of suspended marine macroaggregates can result in diverse and complex assemblages, with specific phyla, such as the CFB, being commonly associated with marine particles. Futhermore, this particle-attached bacterial assemblage was similar to other bacterial assemblages found in marine sediments and terrestrial soils, with respect to the nature of the associated phylogenetic groups.

KEY WORDS: Diversity - Bacteria. Particles. Adriatic Sea

INTRODUCTION tached bacterial communities are often similar to or sometimes lower than those estimated for free-living The occurrence and ecological importance of macro- bacteria. Little is known about the specific bacterial scopic organic aggregates (marine snow) in the pelagic species inhabiting such aggregates (Caron et al. 1986). environment has been demonstrated in a variety of dif- Potential formation of methane (Karl & Tilbrook 1994), ferent coastal and open ocean environments. Marine nitrogen fixation by cyanobacteria (Paerl & Pruffert snow has been found to be enriched in nutrients, trace 1987), sulfide production (Shanks & Reeder 1993), and metals, microbial biomass and production when com- the finding that organic particles may sometimes pared to the surrounding water (Alldredge & Silver exhibit distinct oxygen and pH gradients (Alldredge & 1988, Caron et al. 1986, Herndl et al. 1993). However, Cohen 1987, Paerl 1984) all suggest that particles may biomass specific activities (e.g. ectoenzymatic activity be suitable sites for anaerobic processes in an other- per cell, specific bacterial production) in particle-at- wise oxygenated water column (Sieburth 1991). In addition, comparative phylogenetic evidence based on small subunit ribosomal DNA (SSU rDNA) sequences 'E-mail: [email protected] (DeLong et al. 1993) and low molecular weight RNAs

0 Inter-Research 1998 262 Aquat hlicrob Ecol

(Bidle & Fletcher 1995) of aggregate-associated and (100 pl) contained 2 mM MgC12, 10 mM Tris-HC1 free-living bacteria indicated that particle-attached (pH 8.3), 50 mM KCl, 200 FM deoxynucleotide triphos- bacterial assemblages may differ in composition from phate, 0.5 units Thermus aquaticus DNA polymerase, those inhabiting the surrounding water column. These 0.2 pM of each oligonucleotide primer and 100 ng data also suggest potential differences in the metabolic DNA template. Thermal cycling was performed on a capacities of particle-attached versus free-living Perkin Elmer Thermal Cycler, with denaturation at marine bacterial assemblages. The above observations 95°C for 1.5 min, annealing at 55°C for 1.5 min and suggest that there are substantial microbiological and extension at 72OC for 1.5 rnin for a total of 30 cycles. functional differences between particle-attached and The primers used for amplification of bacterial SSU free-living bacteria. rDNA were (Lane 1991): Intense formation of large marine snow particles, 27F: AGA GTT TGA TCC TGG CTC AG which occurred in the northern Adriatic Sea in summer 1492R: GGT TAC CTT GTT ACG ACT T 1991, has been described in a series of publications Ribosomal DNA cloning, sequencing, and phyloge- which addressed a variety of aspects on the chemical netic analysis. Amplified 16s SSU rDNA fragments and biological properties and formation of marine from 3 independent reactions were pooled and snow in the Adriatic (Bochdansky & Herndl 1992a, b, sequentially extracted using equal volumes of phe- Herndl 1992, Kaltenbock & Herndl 1992, Miiller- nol:chloroform (1:l) followed by a chloroform:isoamyl Niklas et al. 1994, Rath & Herndl 1994). Here we alcohol (24:l) extraction. The DNA products in the describe the phylogenetic diversity of bacteria associ- aqueous extract were ethanol precipitated and resus- ated with marine snow as determined by cloning and pended in 1:10 volume of sterile water. The purified sequencing of SSU rDNAs derived from the complex amplification products were cloned using a cornrner- assemblage. cial 'T-vector' cloning system (Invitrogen Corp, San Diego, CA, USA) following the manufacturer's recommendations. Plasmid minipreparation was performed METHODS by alkaline lysis (Sambrook et al. 1989). Insert-containing clones were identified by the size of the recombi- Sample collection. Marine snow samples were col- nant plasmid and by PCR amplification of the insert lected in the Gulf of Trieste (northern Adriatic Sea) using M13F and M13R primers. Restriction fragment about 2 km off the Laboratorio di Biologia Marina, length polymorphisms (RFLPs) of the amplified inserts Aurisina (Italy), on 11 August 1991, during a period of were used to identify unique rDNA clones. The PCR intense marine snow formation. Samples were col- amplification conditions were identical to those lected by SCUBA divers using 780 m1 hand-made described above, with the following exceptions: plas- plexiglass syringes (rinsed with 0.1 N HCl and distilled mid DNA was added to a final concentration of 1 ng water prior to sampling) at depths between 1 and 15 m. PI-', annealing temperature was 40°C and a total of Aggregate size varied between 5 mm and several cen- 40 cycles was performed. Restriction digests were per- timeters. On the boat, marine snow was transferred to formed following the manufacturer's recomrnenda- a cooling box and held at 4°C. Within 1 h, the samples tions using HaeIII (BRL, Gaithersberg, MD, USA). were centrifuged at 13000 X g for 20 min at 4OC. The Fragmen.ts were separated by agarose gel electro- resulting pellet was divided into 1 g aliquots (wet phoresis using a 3 % (w/v) wide range agarose (Sigma, weight), transferred into Eppendorf vials and stored St. Louis, USA). frozen at -80°C. Sequencing was perform.ed using a commercially Nucleic acid extraction, purification and poly- available sequencing kit and Sequenase 2.0 (USB, merase chain reaction (PCR) amplification. Extraction Cleveland, OH, USA). Universal rDNA sequencing and purification of the nucleic acids were performed primers (Lane 1991) and M13 forward and M13 by gentle enzymatic and detergent lysis, pheno1:chlo- reverse primers were used. Sequences were aligned in roform extraction and CsCl equilibrium density gradi- the mutilple sequence editor, Genetic Data Environ- ent centrifugation as previously described (DeLong ment (GDE 2.2, Smith et al. 1994), and the SSU rDNA 1992, DeLong et al. 1993). Approximately 50 mg wet database available from the ribosomal database pro- weight of marine snow was extracted. Nucleic acid ject (RDP, Maidak et al. 1994). concentrations were estimated by Hoechst dye binding Phylogenetic distance analyses were performed in conferred fluorescence (Hoefer TK), following the GDE using the algorithm of DeSoete (1983). Similarity manufacturer's recommendations. Ribosomal DNA searches were performed using the program 'SIMI- was amplified from purified DNA using GeneAMP Kit LARITY-RANK' on the RDP server (http://rdpgopher. reagents (Perkin Elmer Cetus, Norwalk, CT, USA) as 1ife.uiuc.edu) (Maidak et al. 1994). Full length se- recommended by the manufacturer. Reaction mixtures quences were also screened for potential chimeras Rath et al.: Marine-snow-associated bacterial populations 263

using 'Check-Chimera' (Maidak et al. 1994). Se- sequence GGCC. The fragment patterns of individual quences have been submitted to GenBank and were clones were distinct and readily dinstinguishable. Of assigned the following accession numbers: the 95 characterized clones, 90 different distinguish- AF030782, ADRIATIC15; AF030783, ADRIATIC79; able RFLP types were identified. One RFLP type was AF030784, ADRIATIC84; AF030785, ADRIATIC87; represented 3 times, and 3 patterns were identified AF030786, ADRIATIC83; AF030787, ADRIATIC80; twice in the library. Estimated percent coverage, de- AF030788, ADRIATIC74; AF030790, ADRIATIC92; fined as [l - (n/N)] X 100, where n is the number of AF03079 1, ADRIATIC97; AF030792, ADRIATIC95; unique clones detected in a subsample of size N, ap- AF030793, ADRIATIC64; AF030794, ADRIATICR19; proximates the probability that all species present in a AF030796, ADRIATICG; AF030797, ADRIATIC54; given sample are represented at least once in the sub- AF030798, ADRIATIC31; AF030799, ADRIATIC69; sample (Good 1953, Mullins et al. 1995). RFLP analysis AF030800, ADRIATIC86; AF030801, ADRIATIC81; of the 95 clones in the Adriatic Sea marine snow library AF030802, ADRIATIC77; AF030803, ADRIATIC100; indicated that the coverage was approxin~ately5.3 %. AF030804, ADRIATIC53; AF030805, ADRIATICGO; Partial sequences (approximately 200 bp, positions AF030806, ADRIATIC72; AF030807, ADRIATIC99, 240 to 440, E. coli numbering) were obtained for 41 AF030808, ADRIATIC89; AF030809, ADRIATICR24; randomly chosen clones to initially determine the ap- AF030810, ADRIATIC52; AF030811, ADRIATIC61; proximate phylogenetic affiliation of individual clones. AF030812, ADRIATIC76; AF030813, ADRIATICSO; Initially, the SIMILARITY-RANK option from the RDP AF030814, ADRIATICR27; AF030815, ADRIATICR16; was used to identify the most similar sequences avail- AF030816, ADRIATIC33; AF030817, ADRIATICR20; able in the unaligned database. Two clones with iden- AF030818, ADRIATIC?; AF030819, ADRIATICR25; tical HaeIII restriction fragment patterns were ana- AF030820, ADRIATIC65; AF030821, ADRIATICSO; lyzed to assess their sequence similarity. Clones AF030822, ADRIATIC91. ADRIATIC77 and ADRIATIC100, which have identical Phylogenetic relationships were inferred using the HaeIII restriction patterns, also had identical rDNA se- suite of programs available in PHYLIP (version 3.5; quences over the 200 bp region analyzed. Sequence Felsenstein 1989) on a Sun Sparc 10 workstation. Boot- data derived from clones containing unique HaeIII re- strapped data sets were generated using 'seqboot', and striction patterns indicated that the rDNA clones ana- evolutionary distances calculated from the resulting lyzed were derived from a wide spectrum of phyloge- data sets with 'DNAdist', using the lnlura 2 parame- netically diverse Bacteria. Although high resolution ter model, assuming a transition/transversion ratio of phylogenetic analyses are best perfomed with full 2.0. Neighbor joining analyses on the evolutionary dis- length rDNA sequences, approximate phylogenetic tance data sets were performed using 'neighbor', and placement is possible (and analytically advantageous the results of 100 bootstrap replications compiled using for screening large numbers of clones) using shorter 'consensus'. Phylogenetic trees were graphically visu- regions of rDNA sequence (Schmidt et al. 1991). Phylo- alized using 'treetool', which was obtained by anony- genetic analyses of shorter sequence regions (Table 1, mous ftp from the RDP (Maidak et al. 1994). Figs. 1 & 2a, b) revealed that 8-Proteobacteria (23.1 %,

RESULTS 10 3 To initially assess the diversity of recovered rDNA clones, RFLPs in re- CFB amplified SSU rDNA fragments were 6 - Pro~eobacreria compared (DeLong et al. 1993, Moyer et a - Proleobacreria al. 1996). Various restriction enzymes y - Pro~eohacrer~a (HaeIII, EcoRI, PstI) were tested. As ex- 1 pected, enzymes with 6 bp recognition Gram-pos~r~veEubaclcria sequences revealed only a few differ- E - Prorrobacleria ences between clones. Restriction en- Ctilan~ydia/Ver~comirobium zymes with 4 bp recognition sequences Plancronlyces-brarlch were therefore used to roughly assess clonal diversity. Ninety-five clones of the Fig. 1.Recovery of Adriatic Sea macroaggregate-associated bacterial riboso- rDNA library were character- xnal DNA clones belonging to different phylogenetic groups. The relative per- ized by RFLP analysis using HaeII1, centage of clones affiliated with each qroup,- - out of a total of 39 clones which cleaves the tetrameric recognition sequenced, is indicated 264 Aquat Microb Ecol 14: 261-269, 1998

n = 9) represented the predominant group in the DISCUSSION Adriatic marine snow rDNA library, followed by the Cytophaga-Flavobacteria-Bacteroides (CFB) branch PCR-based studies of phylogenetic diversity are sub- (17.9%, n = 7). All subdivisions of the Proteobacteria, ject to their own inherent errors, biases and artifacts, except for the 0-Proteobacteria subdivision, were rep- as is any methodology applied to study complex envi- resented in the aggregate-attached bacterial rDNA li- ronments or diverse microbial consortia (Liesack et al. brary (Table 1, Figs. 2b & 3). One Adriatic rDNA clone 1991, Reysenbach et al. 1992, hann et al. 1995, out of 39 sequenced was affiliated with Planctomyces Suzuki & Giovannoni 1996, Wang & Wang 1996). and close relatives (Figs. 2a & 3). The other clones Biases and artifacts include the potential creation of were affiliated with the Verrucomicrobium branch, or chimeric molecules during amplification (Liesack et al. the Gram-positive bacteria (Figs. 1 & 2, Table 1). 1991, Robinson-Cox et al. 1995, Wang & Wang 1996), To substantiate preliminary phylogenetic placement overrepresenation of specific groups as a function of based on partial sequence analysis, 10 clones were se- increasing PCR cycle number (Suzuki & Giovannoni lected for more extensive analysis. Analyses of nearly 1996), and underrepresentation due to potential complete rDNA sequences (Fig. 3) generally confirmed primer mismatches. Such potential artifacts suggest the phylogenetic placement within the various bacter- caution in the interpretation of PCR-based data sets, ial phyla inferred from partial sequences (Figs. 2 & 3). particularly in the description of 'novel' taxa, or with Analyses of nearly complete sequences also verified a regard to assumptions about the quantitative repre- close affiliation of the rDNA fragments of clones ADRI- sentation of specific taxa based on PCR-product recov- ATIC76 and ADRIATICR16 with marine sulfate-reduc- eries. Despite these potential problems, the utility of ing bacteria (SRB) of the 6-Proteobacterial subdivision the PCR for microbial diversity studies is still apparent. (Devereux & Mundfrom 1994), the highest similarity For instance, PCR-based analyses of oceanic pico- being shared between these 2 SRB-like sequences and plankton (Fuhrman et al. 1992, Giovannoni et al. 1990) Desulforhopalus vaculolatus. The close phylogenetic have been, supported in parallel studies using genomic relationship of cloned rDNA fragment ADRIATIC87 to shotgun clone libraries (not involving PCR biases) from SSU rDNA from Gram-positive bacteria, and the high similar assemblages (Schmidt et al. 1991). In addition, similarity of ADRIATIC74 to SSU rDNA from members the independent recovery of near-identical rDNA of the Planctomycetales, was also supported in full sequences (98 to 99% similarity) from as-yet unculti- length sequence analyses (Fig. 3). vated bacterial (Giovannoni et al. 1990, Schmidt et al.

Microscilla aggregafis ADRIATIC6 Fig. 2. Phylogenetic analysis of partial ylophugu lyticu sequences (168 nucleotide positions) of -ADRIATIC97 small subunit ribosomal DNA (SSU CFB rDNA) amplified and cloned from macro- Cyrophagasalnronicolor aggregate-associated bacteria from ADRIATIC83 the Adriatic Sea (ADRIATIC) affiliated (A) with the CFB, Gram-positive, Verru- comicrobium and Planctomyces groups PLANCTOMYCETALES and (B)(facing page) with the Proteobac- teria. The trees were inferred by neigh- Plunctomycesmaris bor join~nganalyses using PHYLIP (ver- ADRIATIC74 sion 3.5; Felsenstein 1989). Distances Pirellrrla staleyii were calculated with 'DNAdist' using the env.agg8 PirefluLamanna lmura 2 parameter model assuming a Gemnrnmobscuriglobus transistion/transversionratio of 2.0. Isosphaerapallido Neighbor joining analysis was conducted on 100 bootstrap replicates with random CHLAMYDIAJ taxon addition. Values represent the per- VERRUCOMICROBIUM centage of bootstrap replicates greater than 50% which s1.1pport the assocrated node In the majol'ity rule consensus tree. Scale bar represents the number of fixed mutations per 100 nucleotlde residues. Thermosipho afn'can us Fervidobacte- rium islandicurn and Geotoga subterra- Closlridiumoceanicum nea were used as outgroups. Scale bar Eubacteriurnrenue represents the number of fixed mutations ADRIATIC87 GUM+ per 100 nucleotide residues Closrridiumprmrinolylieum Rath et al.: Marine-snow-associated bacterial populations 265

1991, DeLong et al. 1993, Fuhrman et al. 1993, Mullins century (Degobbis et al. 1995). A massive formation in et al. 1995) and archaeal (DeLong 1992, Fuhrman et al. 1991 was investigated to study the course of various 1992, DeLong et al. 1994) phylotypes indicates that microbial, nutritional and chemical characteristics of many of the novel sequences recovered by these tech- the aggregates and the aggregate-attached communi- niques are genuine and not the result of amplification ties (Bochdansky & Herndl 1992, Kaltenbock & Herndl artifacts. Indeed, rDNA sequences reported for newly 1992, Herndl et al. 1993, Muller-Niklas et al. 1994, cultivated marine oxygenic photoautotrophs (Prochlo- Rath & Herndl 1994). 'Aged' stages of marine snow rococcus marinus) were highly similar to those de- which we used in this study have been described as scribed in prior mixed population PCR analyses 'gel-like aggregates' (Muller-Niklas et al. 1994) or (Schmidt et al. 1991, Urbach et al. 1992, Mullins et al. 'clouds' (Kaltenbock & Herndl 1992). These aggre- 1995). Additionally, phylogenetic hypotheses which gates accumulated at the pycnocline and are charac- originated from analysis of PCR amplified SSU rDNA terized by a massive increase in volume-specific dry have been subsequently supported by analyses of mass. Also typical for these particles is the enrichment highly conserved, rDNA-linked protein encoding of high molecular weight organic material and an genes identified by 'chromosome walking' (Stein et al. enormous enrichment in the bacterial biomass (Miiller- 1996). Niklas et al. 1994). In contrast low molecular weight The formation of marine snow in the northern Adri- dissolved organic matter (i.e. free amino acids) was not atic Sea has been observed and recorded for over a found to be enriched in these aggregates. As with marine snow samples col- lected in the Santa Barbara Channel Pelobacrerpropionicus l0 Archangiumgephyra (CA, USA; DeLong et al. 1993), clones ~Myxococeirsxanthus affiliated with the y-Proteobacteria, Polyangirrmcellrtlosum CFB, and Planctomyces groups were detected in the Adriatic marine snow Bdellovibriosfolpii ADRIATICR27 bacterial rDNA library (Table 1, Desulforhopalusvaruolorus Figs. 1-3). Additionally, several SSU ADRIATICR16 ADRIATIC76 rDNA fragments recovered in Adriatic 6 Sea marine snow were highly similar Desufobulbus.~ro~io~iicus . I 7 Pelobacterace&ler~icus to SSU rDNA sequences from bacteria ADRIATIC~~ with known surface-associated life his- Desulfomoniletiedjei tories (e.g. ADRIATIC? with Hypho- ADRIATICRZO monas jannaschii; ADRIATICG, 54, 79, 80, 83, 92, and 97 with members of the Merhylornonassp. strain 761 S svm. Solenrvavelun~ Cytophaga-Flavobacteria group). In contrast to marine snow samples from . - - - - - . . - . - other studies, however, the phylogenetic diversity was much greater in the bacterial assemblages in Adriatic Murinornorrasvagu marine snow samples (DeLong et al. ADRIATIC69 1993, Fowler & DeLong in unpubl.). Vibriopelagirts Some of the differences between the ANeronronashalo~lanktis different marine snow samples may reflect differences in successional stage (age) of the marine snow and the types of algal species which initially form the aggregates' structural and chemical ADRIATIC72 matrices. The interior of the Adriatic ADRIATIC99 marine snow was highly anaerobic,

Hyphomonasjannaschiana apparently due to the high nutrient r ADRIATIC90 levels and associated high metabolic Hirsclrinbaltica (respiratory) activity, as well as the substantial diffusive barrier provided 59 Arcobaclernkrofigilis I 99 ADFLIATICRZS by the large gel-like organic matrix. G- ADRIATIC50 A nocturnal increase and diurnal ADRIATICIOO - Campylobaclerjejuni decrease of sulfide observed at 266 Aquat Microb Ecol 14: 261-269, 1998

Table 1 Nearest nelghbors and inferredphylogenetic group of 39 partially sequenced clones.CFB: Cytophaga-Flavobacteria- Bacteroides.Sdb: similarity coefficientused by the RDP

Clone Presumptive phylogenetic group Nearest neighbord Sab YO coefficient similarity

ADRIATIC6 CFB Microscilla aggregans subsp.catalatica 0.530 89.5 ADRIATIC7 a-Proteobacteria Hyphomonas sp.str. MHS3 0.767 97.0 ADRIATIC15 y-Proteobacteria Symbiont of Solemya 0.809 88.3 ADRIATIC31 ChlamydiaNerrucomicrobiuni Verrumicrobiumspinosum 0.464 84.7 ADRIATIC33 S-Proteobacteria Desulfosarcina variabilis 2 0.540 83.9 ADRIATIC50 E-Proteobacteria Arcobacterspp. 1 0.654 90.4 ADRIATIC52 a-Proteobacteria Paracoccusalkaliphilus 0.642 90.2 ADRIATIC53 S-Proteobacteria Bdellovibriostolpii str. U12 0.649 81.3 ADRIATIC54 CFB Myroides odoratus 0.729 91.3 ADRIATIC60 6-Proteobacteria Polyanq~umcellulosum 0.461 83.7 ADRIATIC61 y-Proteobacteria Vibnonigripulchrituda 0.543 87.5 ADRIATIC64 Gram-positiveEubacteria Propion1bacterium acnes 0.834 91.9 ADRIATIC65 6-Proteobacteria Desulfuromonaspalmitatus 0.553 84.1 ADRIATIC69 y-Proteobacteria Alteromonasmacleod~i 2 0.751 95.1 ADRIATIC72 a-Proteobacteria DMSP degrading bacteriumstr. LFR 0.800 95.6 ADRIATIC74 Planctomyces branch Pirellula sp. str. Schlesner 1 0.731 90.3 ADRIATIC76 6-Proteobacteria Desulforhopalusvacuola tus 0.754 95.2 ADRIATIC77 E-Proteobacteria Arcobacternitrofigh-s 0.640 93.0 ADRIATIC79 CFB Microscilla furvescensstr. TV-2 0.632 91.0 ADRIATIC80 CFB Porphyromonasendodontalis 0.678 90.3 ADRIATIC81 y-Proteobacteria Methylomonassp. str. 761 0.698 90.9 ADRIATIC83 CFB Cytophaga fermentans 0.580 90.4 ADRIATIC84 Gram-positiveEubacteria Heliobacteriumchlorum 2 0.417 71.9 ADRIATIC86 a-Proteobacteria Roseobacter1itoraLis 0.698 94.9 ADRIATIC87 Gram-positiveEubacteria Eubactenum tenue 0.613 90.0 ADRIATIC89 y-Proteobacteria Symbiont of Lucinoma aequizonatagill 0.627 87.5 ADRIATIC90 a-Proteobacteria Bartonella quintana 0.710 96.3 ADRIATIC91 6-Protoebacteria BdeLlovibnostolpii str. UKi2 0.460 85.2 ADRIATIC92 CFB Flexibacter filiformisstr. FXe l 0.478 86.8 ADRIATIC95 Gram-positiveEubacteria Heljobacteriumchiorum 2 0.367 79.1 ADRIATIC97 CFB Flavobacteriurn salegens 0.763 91.2 ADRIATIC99 a-Proteobacteria Roseobacteralgicola 0.894 99.1 ADRIATIC100 E-Proteobacteria Arcobacternitrofighs 0.659 93 5 ADRIATICR16 6-Proteobacteria Desulforhopalus vacuolatus 0.749 93.4 ADRIATICR19 ChlamydiaNerrucom.icrobium Verrumicroblumspinosum 0.481 79.7 ADRIATICR20 6-Proteobacteria Bdellovibr~ostolpii str. UKi2 0.668 88.6 ADRIATICR24 y-Proteobacteria Methylornonas sp.str. 761 0.707 86.3 ADRIATICR25 E-Proteobacteria Arcobacternitrofigilis 0.554 89.9 ADRIATICR27 6-Proteobacteri.a Syntroph us gentianae 0.716 89.9

'The closest matching sequencefrom a cultivated and characterizedstrain was identifiedusing SIMILARITY-RANK (Maidek et al. 1994). The analysis is based on approximately 200 bp, positions 240 to 440, E. coli numbering. (In some cases, higher similarities were found withenvironmental rDNA clones,or uncharacterized strains.) Unrestrictedpairwise sequence similarities were calculated using the General Data Environmentsoftware package (Smith etal. 1994)

marine thermoclines (Cutter & Krahforst 1988) might snow-associated bacterial rDNAs (Table 1, Fig 2) to indicate that a sulfidogenic bacterial consortium may other isolated or cloned phylotypes occurring in ter- be active in some marine snow habitats. Anaerobic restrial soil (Borneman et al. 1996, Liesack & Stacke- processes on particles accumulating at the pycno- brandt 1992) or marine sediment communities (Dev- cline-the 'transfer of benthic processes to the pycno- ereux & Mundfrom 1994) supports the hypothesis that clinel-was proposed by Sieburth (1991), and pelagic particles may, under suitable conditions, form prompted the formulation of the 'false benthos' con- a 'false benthos' sensu Sieburth (1991). In turn, sedi- cept. Recently, sulfide production in marine snow has ment bacterial communties may be influenced by been directly observed in both laboratory-generated such particle-attached bacteria, since the slnking par- and f~eld-collectedparticles (Shanks & Reeder 1993). ticles ultimately may give rise to the sediments them- The close phylogenetic affiliation of several marine- selves (Novitsky 1.990). Rath et al.- Marine-snow-associated bacterial populations 267

Not suprisingly, our results differ from those of a recent study of freshwater 'lake snow' (Weiss et al. 1996), in which an apparent large proportion ADRIATIC72 of attached bacteria was reported to strain LFR Koscohacferderrifrificans a be affiliated with the 0-Proteobacteria. ADRIATIC86 In contrast, no P-Proteobacteria-like SSU rDNA sequences were detected Rhado.vpir1lurnrubrurn on Adriatic marine snow in this study, Hyphomonosjarinascl~iana ADRlATIC7 nor have they been detected in previ- Hirsclrio bahica ous (DeLong et al. 1993) or ongoing Caulohcler cresenlus surveys (Fowler & DeLong unpubl.) Alterornonas haloplankris of marine macroaggregate-associated Vibrio ~rutriegens Y bacteria. The manifold differences Marinornonos vaga existing between marine and fresh- Cltromatirrm repidurn water systems may explain the differences in the microbial community. Desulfobulbus strain 3prlO Trace nutrient composition and con- ADRIATICR16 centration, phytoplankton species Desulforhopalus vacuolatus ADRIATIC76 from which the macroaggregates are Desrrlfonionile tiedjei derived, salinity, annual temperature ADRIATIC91 variation, available terminal electron Bdellovibrio slolpii acceptors, specific carbon and energy ADRIATIC33 sources, and associated zooplankton species are all expected to be quite different in marine systems. Moreover, the high ion concentration in marine systems facilitates cationic bridging of polysaccharide fibrils (Decho 1990), enhancing the formation of marine Isosphaera pallida snow. Additionally, there are many Cemma!a obscuriglobus 1 ~WQ-a well-documented examples of bacterial species which have specifically Pirellula slaleyli ADRIATIC74 evolved in marine habitats and are not Phnclomyces maris commonly found in freshwater habitats. Therefore, differences in the phy- PLANCTOMYCETALES logenetic composition of bacterial associated with marine Fig. 3. Phylogenetic analysis of the nearly complete sequences of SSU rDNA versus freshwater macroaggregates amplified and cloned from macroaggregate-associated bacteria in the Adriatic are not surprising. Sea. The tree was inferred by neighbor joining analyses with PHYLlP (Felsen- ~t is likely that complex trophody- stein 1989) using 1051 nucleotide positions and neighbor joining boostrap analy- namics (e.g. syntrophic interactions) sis as described in Fig. 2. Values represent the precentage of bootstrap replicates greater than 50% which support the associated node in the majority rule Occur the physical! consensus tree. Scale bar represents the number of fixed mutations per 100 chemical and biological gradients in nucleotide residues. Thermosipho afncanus, Fervidobacteriurn ~slandcurnand marine snow (Alldredge & Silver 1988, Geotoga subterranea were used as outgroups Sieburth 1991, DeLong et al. 1993, Shanks & Reeder 1993). Steep redox- gradients may develop in the particles (Alldredge & blages using similar procedures, appears somewhat Cohen 1987), and their role in habitat structuring has lower than that observed in our Adriatic marine snow been discussed by Paerl (1984). Physical and chemical library, as judged by the greater coverage obtained for gradients, which are presunlably favorable for the similar numbers of clones (Fuhrman et al. 1992, development of highly diverse microbial assemblages, DeLong et al. 1993, Moyer et al. 1994, Hofle & Brettar may form in the complex matrix of marine snow. The 1995, Mullins et al. 1995). The low coverage (5.3 %) phylogenetic diversity found in some other rDNA clone represented in our library may reflect relatively high libraries, obtained from a variety of bacterial assem- phylogenetic diversity which developed in this partic- 268 Aquat Microb Ecol 14. 261-269, 1998

ular particle-attached bacterial community. Similarly Acknor+~ledqements This work was supported In part by the high levels of bacterial diversity have also been Austnan Sclence Foundation (Grant no 7748 to G J H ),by the observed or postulated for soil ecosystems (Torsvik et Styrian Government, by the W~enerHochschul~ubdaumsfond (to J R ), and Unlted Statt.s NSF grants OCE 95-29804 and al. 1990, Borneman et al. 1996), habitats which repre- OPP94-18442 to E F D , O'cR grant N0014-93-1-0731 to E F D , sent similar spatially structured microhabitats with NOAA/West Coast NURC Project no ("493-01 to E F D associated complex physical and chemlcal 'microgra- dients' LITERATURE CITED Assessment of the bulk activity of highly diverse and complex assemblages associated with marine snow Alldredge AL. Cohen Y (1987) Can mcroscale chemical may be extremely difficult, since commonly employed patches persist in the sea? Microelectrode study of marine radiolabeling techniques (thymidine or leucine incor- snow, fecal pellets. Sclence 235589-691. poration) may not be adequate to measure the particle- Alldredge A, Silver M (1988) Charactenstics, dynamlcs and signif~canceof marine snow. Prog Oceanogr 20:41-82 associated metabolic diversity. This may be relevant to Amann RI, Ludwig W, Schleifer KH (1995)Phylogenetic iden- our study, since the CFB Lineage appears to be a fre- tification and In situ detect~onof individual microbial cells quently recovered group in marine, particle-attached without cultivation. Microb~olRev 59(1):142-169 assemblages, and various Flavobacteria isolates have Bidle KD, Fletcher M (1995) Comparison of free-hv~ngand particle-associated bactenal communities in the Chesa- been shown to be unable to incorporate thymidine peake Bay by stable low-molecular-we~ghtRNA analysis. (Davis 1989). 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Editorial responsibility. John Dolan, Submitted: June 23, 1997; Accepted: December 19, 1997 Villefranche-sur-Mer, France Proofs recelved from author(s): March 12, 1998