The Journal of Microbiology (2010) Vol. 48, No. 5, pp.573-585 DOI 10.1007/s12275-010-0151-5 Copyright གྷ 2010, The Microbiological Society of Korea

Bacterial Diversity in the Sediment from Polymetallic Nodule Fields of the Clarion-Clipperton Fracture Zone

Chun-Sheng Wang1,3,4*, Li Liao2, Hong-Xiang Xu5, Xue-Wei Xu3,4, Min Wu2, and Li-Zhong Zhu1

1College of Environment and Resource Sciences, Zhejiang University, Hangzhou 310058, P. R. China 2 College of Life Sciences, Zhejiang University, Hangzhou 310058, P. R. China 3 Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration, Hangzhou 310012, P. R. China 4 Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012, P. R. China 5 College of Life Sciences, Wenzhou Medical College, Wenzhou 325035, P. R. China (Received April 19, 2010 / Accepted June 21, 2010)

The Clarion-Clipperton Fracture Zone (CCFZ) is located in the northeastern equatorial Pacific and contains abundant polymetallic nodules. To investigate its bacterial diversity, four libraries of 16S rRNA genes were constructed from sediments of four stations in different areas of the CCFZ. In total, 313 clones sequenced from the 4 libraries were assigned into 14 phylogenetic groups and 1 group of 28 unclassified bacteria. High bacterial diversity was predicted by the rarefaction analysis. The most dominant group overall was Proteobacteria, but there was variation in each library: Gammaproteobacteria was the most dominant group in two libraries, E2005-01 and ES0502, while Alphaproteobacteria and Deltaproteobacteria were the most dominant groups in libraries EP2005-03 and WS0505, respectively. Seven groups, including Alphaproteo- bacteria, Gammaproteobacteria, Deltaproteobacteria, Betaproteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, were common to all four libraries. The remaining minor groups were distributed in libraries with different patterns. Most clones sequenced in this study were clustered with uncultured bacteria obtained from the environment, such as the ocean crust and marine sediment, but only distantly related to isolates. Bacteria involved in the cycling of metals, sulfur and nitrogen were detected, and their relationship with their habitat was discussed. This study sheds light on the bacterial communities associated with polymetallic nodules in the CCFZ and provides primary data on the bacterial diversity of this area. Keywords: CCFZ, sediment, bacterial diversity, 16S rRNA gene, polymetallic nodule

The Clarion-Clipperton Fracture Zone (CCFZ) located in the China Ocean Mineral Resources R & D Association northeastern equatorial Pacific is considered one of the most (COMRA) has a contract area for exploration of polymetallic commercially important nodule areas of the World Ocean nodules in the CCFZ that covers about 7.5u104 km2, including (Thiel, 2001). Polymetallic nodules are also known as two regions (east and west regions). The microbial diversity manganese nodules or ferromanganese nodules because of near the west region of the COMRA’s contract area has been their high manganese and iron content and the concentration studied using culture-dependent and -independent methods of other heavy metals, such as copper and nickel (Margolis (Xu et al., 2005). Six phyla have been detected in bacterial and Burns, 1976). They can be found in many regions in the communities, with a dominance of Gammaproteobacteria. vast ocean at water depths ranging from 4,000 to 6,000 m, but However, few investigations have been done on the microbial they mainly aggregate in CCFZ areas as mature and large diversity in other Chinese claim areas of the CCFZ. Here we nodules (Glasby et al., 1982). The mechanism of formation of studied the microbial diversity of four stations in the CCFZ by manganese nodules is as yet unclear, but the function of constructing 16S rRNA gene libraries, with the hope of microbes and the impact of physical and chemical factors in discovering special bacterial communities related to the abyssal environment are critical (Wang and Müller, 2009; manganese nodules. Wang et al., 2009a, 2009b). Some iron- and manganese- Because of the commercial and strategic interests in marine oxidizing/reducing bacteria have been isolated, such as polymetallic nodules, deep-sea mining has great potential in Shewanella, Hyphomicrobium, Leptothrix, and Pseudomonas. the future. However, mining has some impact on the primary In addition, some direct evidence has been provided using the environment by disturbing the original ecosystem and changing detection of exolithobiontic and endolithobiontic microbial the physical and chemical conditions (Alexis et al., 2006). Due biofilms in manganese nodules to demonstrate their biogenic to the great economic potential of metal exploitation in the origin (Wang et al., 2009a, 2009b). Therefore, it is predicted CCFZ, it is very important to understand the natural microbial that bacteria that actively participate in metal cycling may be diversity in this area before mining. A previous study has found or even be dominant in the sediments from the CCFZ. suggested that the environment requires a very long term to recover from disturbance, making us think before leaping into deep-sea mining (Alexis et al., 2006). Thus, investigation into * For correspondence. E-mail: [email protected]; Tel: +86-571-8196- 3205; Fax: +86-571-8807-1539 the natural microbial diversity of the CCFZ is necessary. In 574 Wang et al.

from 0-10 cm were mixed for each of three stations, WS0505, ES0502, and E2005-1, while sediments from 0-22 cm were mixed for station EP2005-03. The average biogeochemical properties of the sediment samples were determined using energy dispersive X-ray fluorescence (EDXRF) for major and trace elements.

Total DNA extraction and purification Samples were thawed overnight at 4°C before DNA extraction. Two different methods were used to extract DNA from sediment, i.e., using the FastDNA Spin kit for soil (BIO101, USA) and following the established method of (Krsek and Wellington, 1999) with some modifications. In detail, 1 g of sediment sample (wet weight) was Fig. 1. Map of the four stations sampled in this study mixed with 2 ml of DNA extraction buffer [100 mM Tris-HCl, 100 mM

Na2-EDTA, 100 mM phosphate-buffered solution, 1.5 M NaCl, and this study, we surveyed the primary composition of bacterial 1% cetyltriethylammnonium bromide (CTAB), pH 8.0] and treated communities of four stations in the CCFZ for the first time. three times with liquid nitrogen and boiling water consecutively. This study serves as a scientific basis for the selection of Lysozyme was added at a final concentration of 50 mg/ml, the sample environmental reference zones for deep-sea mining and as a was incubated for 1 h at 37°C with shaking at 120 rpm and proteinase primary study of bacterial diversity in northeast Pacific K was then added at a final concentration of 100 mg/ml under the polymetallic nodule fields. same incubation conditions. Next, SDS was added at a final concent- ration of 2% and incubated for 1 h at 60°C. The supernatant was Materials and Methods collected using centrifugation for 1 min at 12,000×g. The precipitate was re-extracted as follows: 1 ml of DNA extraction buffer and 200 Pl Sample collection of 20% SDS were added to the precipitate and incubated for 10 min Deep-sea sediments of the four stations were collected using a TV at 60°C, and the supernatant was collected using centrifugation for 1 multicorer from a CCFZ polymetallic nodule province by R/V “DA min at 12,000×g. Then, 0.6 volume of isopropanol was added for YANG YI HAO” in August 2005. The sampling stations are precipitation for 1 h, and the sample was then centrifuged for 30 min characterized in Table 1 and Fig. 1. Core samples of sediment thicker at 16,000×g. The precipitate was washed with 70% ethanol and dried than 10 cm were divided into 10 1-cm sections after collection. at room temperature before being dissolved in 50 Pl of TE buffer. The Subsamples were put into 10-ml sterile tubes and stored at -20°C until crude DNA extract was purified using a PCR Clean-up kit (QIAGEN, laboratory analysis. To investigate the microbial diversity, sediments USA).

PCR amplification Table 1. Characteristics of the sampling stations The total DNA extracted using the two different methods described Parameters Stations above was amplified. PCR was performed using primers 27F (5ƍ- WS0505 ES0502 E2005-01 EP2005-03 AGAGTTTGATCCTGGCTCAG-3ƍ) and 1492R (5ƍ-GGTTACCTTG TTACGACTT-3ƍ) (Webster et al., 2003). The 50 Pl PCR reaction Latitude (N) 10°2.3c 8°22.5c 8°00c 9°00c system contained 5 Pl of 10u buffer, 0.2 mM dNTPs, 20 pM of each Longitude (W) 154°0.9c 145°23.8c 145°00c 120°25c primer and 1 U of Taq DNA polymerase. The PCR program was Depth (m) 5120 5307 5197 4299 performed as follows: denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 75 sec and a pHa 7.78 7.80 ND 7.82 final extension at 72°C for 7 min. PCR products were analyzed using DO ( M)a 360 315 ND 330 ȝ 1% agarose gel electrophoresis and recovered using a gel recovery kit DIP (ȝM)a 2.55 2.53 ND 2.50 (QIAGEN). The recovered DNA products from the two different DIN (ȝM)a 22.31 45.00 ND 45.07 extraction methods were mixed for each sampling station to construct 16S rRNA gene libraries. TP (ȝM)a 2.94 3.93 ND 2.92 a TN (ȝM) 39.23 46.92 ND 33.07 Library construction and sequencing b SiO2 (%) 50.83 53.33 54.68 ND The recovered PCR products were ligated into pMD18-T vectors b (TaKaRa, Japan) at 16°C for 6 h and then transformed into competent Al2O3 (%) 11.13 11.72 11.43 ND b Escherichia coli TOP10 cells. Four libraries of 16S rRNA genes were Fe2O3 (%) 6.77 5.36 5.57 4.15 constructed from the four sampling stations, respectively: EP2005-03, b CaO (%) 1.61 0.91 1.04 ND E2005-01, ES0502, and WS0505. Positive clones were selected using MnO (%)b 1.33 0.29 0.47 0.09 the blue-white selection method and randomly sequenced using M13 Type of Silicon Silicon Silicon Calcic and primers on an ABI 377 DNA sequencer. sediment clay clay clay Silicon clay Rarefaction analysis and phylogenetic analysis a Parameters of near-bottom water, which are the average values of a 50-m water column near the sea floor. Sequences were checked with the CHECK_CHIMERA program in b Parameters of sediments. ND, not determined; DO, dissolved oxygen; DIP, the Ribosomal Database Project (RDP) to exclude possible chimeras dissolved inorganic phosphorus; DIN, dissolved inorganic nitrogen; TP, total (Maidak et al., 2001). The remaining sequences were assigned into phosphorus; TN, total nitrogen. Bacterial diversity associated with polymetallic nodules 575G

120 Results

100 E2005-01 Rarefaction analysis and phylogenetic analysis EP2005-03 In total, 313 sequences were obtained after excluding chimeras, ES0502 80 including 67, 115, 63, and 68 sequences in libraries E2005-01, WS0505 1:1 reference EP2005-03, ES0502, and WS0505, respectively. The 313 60 sequences were assigned into 223 OTUs (•97% similarity), including 45, 71, 58, and 49 in libraries E2005-01, PE2005-03, No. of OTUs No. 40 ES0502, and WS0505, respectively. Rarefaction curves of the four libraries were constructed based on the observed OTUs 20 for each library (Fig. 2). Compared with the 1:1 reference curve, rarefaction curves of the four libraries had smaller 0 slopes but indicated high bacterial diversity. Although 0 20 40 60 80 100 rarefaction curves did not reach a plateau, they did reveal the No. of clones relative diversity of the four libraries; library ES0502 had the highest diversity, while library E2005-01 had the lowest Fig. 2. Rarefaction curves of the four bacterial libraries. The 1:1 diversity. reference curve indicates that each sequenced clone belongs to a The 4 libraries contained 14 phylogenetic groups and 28 different OTU. unclassified bacteria (Fig. 3). The most dominant group was Alphaproteobacteria, followed by Gammaproteobacteria, operational taxonomic units (OTUs) at a level of sequence similarity accounting for 23.3% and 21.7% of the 313 sequences, •97%, and rarefaction curves were calculated using the Distance- respectively. The dominant non-Proteobacteria group was Based OTU and Richness (DOTUR) program (Schloss and Acidobacteria, which accounted for 7% (22) of the total Handelsman, 2005; Liao et al., 2009). All of the OTUs were classified sequences. Betaproteobacteria, Chloroflexi, Firmicutes, and compared using the RDP. Relatives were downloaded for the Verrucomicrobia, and candidate division TM6 were minor construction of phylogenetic trees using MEGA software version 4.0 groups, with ”9 sequences each. Phylogenetic trees of the 14 using the neighbor-joining method with the Kimura 2-parameter phylogenetic groups and unclassified bacteria were constructed model at the bootstrap value of 1000 (Tamura et al., 2007). to show the phylogenetic relationships with close relatives (Figs. 4-9). Most of the relatives were uncultured bacterial Sequence accession numbers clones from marine environments, such as deep-sea sediments All 16S rRNA gene sequences obtained in this study were deposited and the ocean crust, whereas only a few clones obtained from in the GenBank nucleotide sequence databank under the accession the present study could be related to isolates. numbers GU983390 to GU983612.

45

40

35 E2005-01E2005-01 EP2005-03EP2005-03 30 ES0502ES0502 25 WS0505WS0505

20

15 No.of library a in clones 10

5

0 a a a s s d a ri i i es e tes ira e i e er eria er p i M6 t t teria cut de f T c c ct ycet ssi bac ba roidete mi tros a icrob on o e ir om i cl m eo e eoba noba ct F N isi ot t t Chloroflexi timona un v ro ro Acidobact Ba anct a apr ap p Acti l m haproteobacteria t ta P p m el m Verruco m Be idate di Al a D Ge d G Phylotypes Can

Fig. 3. Phylotype distribution of the four libraries. 576 Wang et al.

Alphaproteobacteria while OTU WS0505b15 only matched the distant clone 004A5 Alphaproteobacteria from library PE2005-03 comprised 14 from sediment of the Northern Bering Sea. OTUs with 39 clones, accounting for the largest percentage of Alphaproteobacteria in the four libraries. Three clusters were Acidobacteria, Actinobacteria, and Nitrospira identified in Alphaproteobacteria, including Rhodospirillaceae, As the biggest non-Proteobacteria group, Acidobacteria (Fig. Hyphomicrobiaceae, and Marinovum (Fig. 4). In addition, four 7) contained six distinct subgroups, including Gp10, Gp21, unknown clusters, named cluster 1 to cluster 4, were detected Gp22, Gp5, Gp9, and Gp6. As of 2007 (Barns et al., 2007), the that were unrelated to any cultures. The most frequently Acidobacteria phylum contained 26 subgroups or 26 genera sequenced OTU was EP2005-03a27, with 20 closely related recognized in the RDP taxonomic hierarchy. Most of these clones grouped into cluster 4. In Rhodospirillaceae, two OTUs subgroups were uncultured Acidobacteria obtained from were found to be associated with isolates Pelagibius litoralis various environments, including metal-contaminated sediment, strain CL-UU02T and Skermanella sp. Ph-4-1. In Hyphomi- acidic drainage, and other environments. All of the six crobiaceae, clones were most closely related to the uncultured subgroups detected in this study were affiliated with uncultured clone EPR3970-MO1A-Bc67 from the ocean crust, except clones from the ocean crust, deep-sea hydrothermal regions, OTU EP2005-03a33, and formed a sister branch of genera and the South-Atlantic Ocean. Pedomicrobium and Hyphomicrobium. In Marinovum, OTU OTU ES0502c21 in Actinobacteria shared 90% similarity EP2005-03b35 shared 96% identity with Marinovum sp. with Solirubrobacter sp. BXN5-15, forming a deep branch in YCSA81. The remaining clones were related to uncultured the phylogenetic tree (Fig. 7). The remaining Actinobacteria bacteria from marine environments, such as marine basalts, clones were associated with uncultured relatives from the the Pacific nodule province, the South-Atlantic Ocean and ocean crust, the Pacific nodule province, soil and the South- hydrothermal regions. Atlantic Ocean, except OTU EP2005-03b29, which formed a sole branch showing very low identity with others. Gammaproteobacteria Nitrospira formed a sister branch with Actinobacteria in the Gammaproteobacteria were the second dominant group with phylogenetic tree (Fig. 7). Half of the clones belonging to 44 OTUs, the maximum found in all the groups. Pseudomonas, Nitrospira were closely clustered with clone E75 from Ectothiorhodospiraceae, and Legionellales were detected with sediment of the Pacific nodule province and clone cultured relatives (Fig. 5). Clones grouped into Pseudomonas P0X3b1A09 from the ocean crust, while the remaining half were most similar to uncultured clones from the ocean crust was associated with clones from the South-Atlantic Ocean. and were closely related to the exclusive isolated relative Pseudomonas sp. NB1-h. Clones belonging to Ectothiorhodos- Planctomycetes and minor groups piraceae were clustered with uncultured bacteria from the Figure 8 shows the phylogenetic relationships of Plancto- ocean crust and the South-Atlantic Ocean and formed a sister mycetes and other minor groups, including Bacteroidetes, branch with isolated strains. Three OTUs in Legionellales Chloroflexi, Verrucomicrobia, and candidate division TM6. shared low similarities with each other and were distantly Planctomycetes formed a large branch compared with other related to the cultured strains in this group. In the four minor groups. The OTU EP2005-03c8 (with two related unidentified clusters (clusters 1-4), most clones shared high clones) was identical with clone Ucd1519 from the South- similarities (•98%) with relatives retrieved from the South- Atlantic Ocean. A few clones were related to uncultured Atlantic Ocean. Cluster 3 was characterized by relatives of relatives from non-marine environments, including mangrove endosymbionts from marine animals. Cluster 4 was the most soil and microbial fuel cells. However, clones belonging to diverse, containing isolates from different orders. Planctomycetes were not found in library WS0505. Cytophaga sp. NB1-m was the only isolate detected in Bacteroidetes, Deltaproteobacteria and Betaproteobacteria sharing 98% similarity with OTU EP2005-03c30. The Library WS0505 comprised 12 OTUs with 18 clones belonging remaining clones were related to uncultured bacteria from to Deltaproteobacteria, which was more than the other three diverse environments, including marine basalts, marine libraries. Nitrospinaceae, Myxococcales, Syntrophobacterales, sediments, an iron oxide chimney-like structure and biofilms. and Desulfuromonadales were detected within Deltaproteo- Only two OTUs (WS0505b19 and WS0505c24) from library bacteria as well as two unidentified clusters (Fig. 6). All of the WS0505 were grouped into candidate division TM6, a unique OTUs were closer to uncultured bacterial relatives than characteristic of library WS0505. Likewise, two OTUs, cultured strains. However, the only OTU, ES0502c4, in ES0502c14 and ES0502c20, were found exclusively in library Myxococcales was distantly associated with Chondromyces ES0502. Chloroflexi was divided into two separate branches pediculatus strain Cm p17 and had no uncultured relative. The with high bootstrap values and was not found in library Desulfuromonadales group was the largest in the four ES0502. identified groups, with the closest isolates Geoalkalibacter subterraneus strain Red1 and Desulfuromonadales bacterium Firmicutes, Gemmatimonadetes, and unclassified bacteria Tc37 as the outgroup within Desulfuromonadales. Firmicutes and Gemmatimonadetes were not found in Two OTUs, WS0505b15 and ES0502a26, were distantly libraries PE2005-03 and E2005-01, respectively. OTU branched from other clones of Betaproteobacteria (Fig. 6), ES0502c8 showed low similarity with the other clones of which were almost identical with clones from the South- Firmicutes and was distantly related to cultured strains (Fig. Atlantic Ocean. OTU ES0502a26 shared high similarity with 9). The remaining three OTUs of Firmicutes were clustered clone YJQ-2 obtained from a microbial mat in a hot spring, with uncultured clones and distinctly apart from isolates Bacterial diversity associated with polymetallic nodules 577G

68 EP2005-03c4(3) 86 ES0502a3 52 clone JdFBGBact 41(DQ070828)[endolithic microbes in marine basalts] clone E28(AJ966596)[sediment from Pacific nodule province] 98 WS0505b25(3) 95 63 clone HCM3MC83 6D FL(EU373881)[Eastern Mediterranean Sea sediment] E2005-01a2(2) EP2005-03c5 97 99 EP2005-03a4 Pelagibius litoralis strain CL-UU02T(DQ401091) 60 79 ES0502a2 100 clone Ulr15112(AM997443)[South-Atlantic Ocean sediment] E2005-01b10 100 clone Ucc1486(AM997985)[South-Atlantic Ocean sediment] Rhodospirillaceae magnetite-containing magnetic Vibrio strain MV1(L06455) clone A4F(FJ205178)[deep-sea hydrothermal region sediment] 70 clone SPG12_60_70_B41(FJ746295)[marine sediment] 79 100 WS0505b32 E2005-01c24 55 EP2005-03b10(2) 94 65 EP2005-03c10 EP2005-03c11 84 ES0502c13(2) 100 WS0505c6(2) 65 clone Ucn1513(AM997413)[South-Atlantic Ocean sediment] Alphaproteobacteria EP2005-03c35 87 Skermanella sp. Ph-4-1(GU195650) E2005-01a5(5) 70 WS0505a21 99 clone EPR3970-MO1A-Bc67(EU491654)[ocean crust] EP2005-03b4(3) Hyphomicrobiaceae 97 Hyphomicrobium sp. Ellin112(AF408954) 52 Pedomicrobium manganicum strain ACM 3038(X97691) 85 Hyphomicrobium sp. WG6(AY934488) EP2005-03a33 Phyllobacteriaceae bacterium AMV1(FN297835) Rhodopseudomonas julia strain DSM 11549T(AB087720) Parvibaculum sp. P31(FJ182044) 100 WS0505c23 clone Ulr1513(AM997448)[South-Atlantic Ocean sediment] 99 E2005-01a8 EP2005-03c38 100 E2005-01a24 100 Cluster 1 clone P9X2b3C05(EU491110)[ocean crust] 50 clone Ksed19(EU035887)[coral reef sediment] 50 clone P9X2b7G10(EU491212)[ocean crust] 50 EP2005-03a15(2) 100 ES0502b5 96 WS0505a42 clone SPG12_343_353_B100(FJ746084)[marine sediment] Cluster 2 96 WS0505c21 99 clone ss1_B_01_63(EU050755)[Arctic sedimemt] E2005-01b19 WS0505a5(3) 70 100 clone Ucn1486(AM997422)[South-Atlantic Ocean sediment] Cluster 3 EP2005-03b5 ES0502b20 ES0502b10 clone 125D68(EU735029)[Northern Bering Sea sediment] 100 Cluster 4 97 EP2005-03a27(20) 78 ES0502a9 Albidovulum sp. S1K1(FJ222605) 82 EP2005-03b35 89 Marinovum sp. YCSA81(GQ246213) Marinovum 100 ES0502c3 100 clone I2A(FJ205314)[deep-sea hydrothermal region sediment] 0.01

Fig. 4. Phylogenetic tree of Alphaproteobacteria from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. 578 Wang et al.

77 clone EPR3970-MO1A-Bc21(EU491607)[ocean crust] 52 EP2005-03a32(3) WS0505a17 98 E2005-01a32(3) ES0502a7 96 Pseudomonas sp. NB1-h(AB013829) 77 clone P0X3b5H10(EU491400)[ocean crust] Pseudomonas 75 ES0502a12(2) WS0505c31 94 90 clone EPR3968-O8a-Bc7(EU491736)[ocean crust] 99 EP2005-03c1 E2005-01c4 99 WS0505b35 53 EP2005-03b37(5) 65 WS0505a27(3) 81 clone Ucm15693(AM997395)[South-Atlantic Ocean sediment] 99 ES0502b3(3) E2005-01b8(3) 99 clone Ucn1569(AM997418)[South-Atlantic Ocean sediment] EP2005-03c34 99 Cluster 1 clone EPR3970-MO1A-Bc84(EU491671)[ocean crust] 99 WS0505a32 56 E2005-01b24 98 clone Ucb15704(AM997798)[South-Atlantic Ocean sediment] 99 E2005-01a14 70 WS0505b4 80 Thiohalomonas denitrificans strain HLD 14(EF117913) EP2005-03a38 99 clone Ucp1573(AM997617)[South-Atlantic Ocean sediment] EP2005-03b20 Cluster 2 clone Ucm1583(AM997391)[South-Atlantic Ocean sediment] 99 99 ES0502b25 79 clone Ulr1572(AM997451)[South-Atlantic Ocean sediment] 99 WS0505b7(2)

Ridgeia piscesae sulfur-oxidizing endosymbiont(U77480) Gammaproteobacteria 99 clone P9X2b7A09(EU491165)[ocean crust] EP2005-03c2(2) 99 clone UncDeep7(AM997509)[South-Atlantic Ocean sediment] 99 ES0502a20 Cluster 3 Olavius ilvae Gamma 3 endosymbiont clone OilvEIV1jw2-13(AJ620499) clone 048E82(EU925855)[Northern Bering Sea sediment] E2005-01c20 99 99 EP2005-03c13 67 WS0505c35 Ectothiorhodosinus mongolicus M9T(AY298904) Thioalkalivibrio thiocyanodenitrificans strain ARhD(AY360060) 51 clone P0X3b5C05(EU491387)[ocean crust] 99 WS0505a18 EP2005-03b13 EP2005-03b19 98 clone Ucm1559(AM997285)[South-Atlantic Ocean sediment] 99 ES0502a4(2) Ectothiorhodospiraceae E2005-01b12(3) clone P0X4b2H02(EU491489)[ocean crust] 96 95 EP2005-03b31(2) 99 clone Ucb15511(AM997844)[South-Atlantic Ocean sediment] 83 ES0502a5 58 WS0505b38(2) 73 E2005-01a9 E2005-01a1 96 Coxiella burnetii strain 607(D89792) Legionella sp. OI36(AB058917) Legionellales clone II10B(FJ205344)[deep-sea hydrothermal region sediment] 99 ES0502c9 99 clone CS051(AY279072)[cold-seep sediment] 65 EP2005-03c29 Marinobacterium sp. KW44(FJ716699) 99 Marinomonas blandensis MED 121T(DQ403809) 58 E2005-01c12 99 clone EPR4059-B2-Bc88(EU491588)[ocean crust] 72 EP2005-03c27 Cluster 4 clone P13-13(EU287106)[Pacific Arctic sediment] 90 99 EP2005-03c24 81 E2005-01a18(2) 0.01 99 ES0502c12

Fig. 5. Phylogenetic tree of Gammaproteobacteria from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. Bacterial diversity associated with polymetallic nodules 579G

99 ES0502a13 clone HCM3MC78_10H_FL(EU373914)[Eastern Mediterranean Sea sediment] ES0502b14

clone JTB36(AB015242)[cold-seep area sediment] Cluster 1 90 WS0505a3 clone EPR3965-I2-Bc9(EU491889)[ocean crust] 99 50 WS0505c4 66 clone P9X2b2H03(EU491265)[ocean crust] WS0505c32(3) 99 clone HCM3MC78_4H_FL(EU373913)[Eastern Mediterranean Sea sediment] 100 Nitrospina gracilis strain Nb-3(L35503) Nitrospinaceae Nitrospina sp. 3005(AM110965) ES0502c11 99 98 clone Ucb15382(AM997835)[South-Atlantic Ocean sediment] 100 WS0505a29(3) 99 clone Ucp1528(AM997673)[South-Atlantic Ocean sediment] 100 WS0505a12 99 clone Ucm1579(AM997311)[South-Atlantic Ocean sediment] WS0505a25(3) 100 clone EPR4059-B2-Bc20(EU491531)[ocean crust] WS0505c34 100 clone MSB-5A9(DQ811823)[mangrove soil]

E2005-01c9 Cluster 2 100 clone Ucc15712(AM997956)[South-Atlantic Ocean sediment] WS0505c8

95 clone I1C(FJ205323)[deep-sea hydrothermal region sediment] Deltaproteobacteria 100 WS0505b18 clone SPG12_213_223_B51(FJ746219)[marine sediment] E2005-01c8 99 71 WS0505c25 93 clone Ucm15772(AM997396)[South-Atlantic Ocean sediment] ES0502c4 Myxococcales Chondromyces pediculatus strain Cm p17(AJ233940) ES0502b8 Syntrophobacterales 100 clone SPG12_343_353_B84(FJ746160)[marine sediments] clone Ucm1578(AM997342)[South-Atlantic Ocean sediment] Desulfoacinum infernum strain BalphaG(L27426) 68 ES0502b18 clone Ulrdd 14(AM997480)[South-Atlantic Ocean sediment] 100 ES0502a17 98 clone HCM3MC78_10D_FL(EU373910)[Eastern Mediterranean Sea sediment] 100 Geoalkalibacter subterraneus strain Red1(EU182247) Desulfuromonadales bacterium Tc37(AB260047) 98 ES0502a6 clone AO30(FJ358878)[marine reef sandy sediment] Desulfuromonadales 50 WS0505a6 clone A4C(FJ205191)[deep-sea hydrothermal region] 99 E2005-01c11 63 clone Tomm05_1274_3_Bac121(FM179874)[methane seeps sediment] E2005-01b14 clone I6E(FJ205325)[deep-sea hydrothermal region sediment] clone Ucp15494(AM997682)[South-Atlantic Ocean sediment] EP2005-03b39 100 E2005-01a11 65 EP2005-03b32 56 59 WS0505a20 clone Ucb1581(AM997872)[South-Atlantic Ocean sediment] 100 WS0505b15

clone 004A5(EU734913)[Northern Bering Sea sediment] Betaproteobacteria 100 ES0502a26 52 clone YJQ-2(AY569280)[hot spring microbial mat] 88 WS0505b1 99 clone Ucs1533(AM997697)[South-Atlantic Ocean sediment] E2005-01b13(2) 100 EP2005-03c31(3) 60 clone Ucm1551(AM997379)[South-Atlantic Ocean sediment] ES0502a8 0.01 clone Ucm1550(AM997326)[South-Atlantic Ocean sediment]

Fig. 6. Phylogenetic tree of Betaproteobacteria and Deltaproteobacteria from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. 580 Wang et al.

64 E2005-01c1 100 WS0505b26 EP2005-03b34 76 66 clone PR4059-B2-Bc40(EU491546)[ocean crust] E2005-01a21 100 clone P7X3b4A08(EU491057)[ocean crust] EP2005-03a3 63 77 ES0502a10 clone E17(AJ966591)[sediment from Pacific nodule province] 100 clone P9X2b7C03(EU491180)[ocean crust]

E2005-01c5 Actinobacteria 96 EP2005-03a28 EP2005-03a7 100 WS0505a41 99 clone Ucb1539(AM997746)[South-Atlantic Ocean sediment] 91 EP2005-03a23(2) 95 69 ES0502c7 96 clone UncDee20(AM997522)[South-Atlantic Ocean sediment] 95 86 WS0505a33 69 clone Ucm1542(AM997358)[South-Atlantic Ocean sediment] EP2005-03b29 78 ES0502a21 98 clone AK1DE1_05F(GQ396980)[soil] Solirubrobacter sp. BXN5-15(EU332825) 100 ES0502c21 99 clone P0X4b3G08(EU491455)[ocean crust] ES0502a14 100 WS0505b8(2) EP2005-03b16 87 clone E75(AJ966604)[sediment from Pacific nodule province] EP2005-03c33 Nitrospira 96 clone P0X3b1A09(EU491363)[ocean crust] 100 100 EP2005-03a25(2) clone Ucb15512(AM997842)[South-Atlantic Ocean sediment] ES0502b9 74 100 clone Ucm15565(AM997344)[South-Atlantic Ocean sediment] 79 WS0505c11 100 ES0502a28 99 clone Ucb15518(AM997911)[South-Atlantic Ocean sediment] 100 EP2005-03c39 71 clone P7X3b4C02(EU491064)[ocean crust] Gp10 ES0502a30 100 clone KZNMV-30-B52(FJ712615)[Gas Hydrate sediments] ES0502b2 98 clone B78-61(EU287025)[Pacific Arctic surface sediment] 99 clone ESB2(EF061216)[sediment from Pacific nodule province] E2005-01b29(6) 100 98 clone 26(FJ024305)[marine basalts] 99 Gp21 clone HCM3MC80_5E_FL(EU373941)[canyon and slope sediment] 80 97 E2005-01c10 71 clone B110(AY375083)[western Pacific Warm Pool sediment] EP2005-03c14(2) Acidobacteria 100 clone Creta1-H03(AY533898)[eastern Mediterranean Sea sediment] 100 E2005-01c29 clone HCM3MC78_11E_FL(EU373925)[eastern Mediterranean Sea sediment] EP2005-03b38 Gp22 100 clone 067C77(EU734973)[northern Bering Sea sediment] 100 EP2005-03b18 Gp5 clone Ucm1546(AM997299)[South-Atlantic Ocean sediment] 56 ES0502a19 77 clone P0X4b2E11(EU491480)[ocean crust] Gp9 99 EP2005-03b6 EP2005-03a34 EP2005-03b11 99 WS0505c22 99 clone I7E(FJ205362)[deep-sea hydrothermal region] Gp6 99 clone P0X3b5A04(EU491382)[ocean crust] ES0502c23 0.01 57 89 WS0505c3

Fig. 7. Phylogenetic tree of Acidobacteria, Actinobacteria, and Nitrospira from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. Bacterial diversity associated with polymetallic nodules 581G

78 clone Ucn1533(AM997415)[South-Atlantic Ocean sediment] 96 ES0502c17 EP2005-03b30 clone JdFBGBact 34(DQ070826)[endolithic microbes in marine basalts] 100 98 E2005-01a10(2) clone 065F30(EU925864)[northern Bering Sea sediment] 100 WS0505a16 Cytophaga sp. NB1-m (AB013834) 100 EP2005-03c30 clone A23(GQ249487)[anaerobic marine sediments] Bacteroidetes EP2005-03a5 61 100 75 clone AV19F34b(FJ905636)[iron oxide chimney-like structure] 72 E2005-01a12 96 clone TDNP_Bbc97_188_1_50(FJ516771)[biofilm] 89 WS0505b37 93 clone 35(FJ024309)[marine basalts] 100 EP2005-03b23 clone Ucn1548(AM997417)[South-Atlantic Ocean sediment] 100 ES0502c28 99 clone P0X4b2C08(EU491475)[ocean crust] 100 WS0505b19 Candidate division TM6 clone BD72BR121 (GU363043)[South China Sea sediment] 99 WS0505c24 100 clone S26-95 (EU287395)[Pacific Arctic Ocean sediments] E2005-01a20 100 clone SPG12_343_353_B54 (FJ746136)[ocean sediment] 100 WS0505c9 EP2005-03a37 Chloroflexi clone MD2902-B4(EU048611)[Xisha Trough sediment] 100 clone SPG12_461_471_B32(FJ746327)[marine sediment] 69 WS0505a43(2) 99 clone Tun3b.F1(FJ169213)[symbiont] ES0502c14 Verrucomicrobia 99 clone SPG12_60_70_B71(FJ746320)[marine sediments] 100 ES0502c20 54 clone E50 (AJ966598)[Pacific nodule province] 61 E2005-01a7 100 clone LC1-9(DQ289908)[permeable shelf sediments] 100 EP2005-03c41 clone Ucm1561(AM997286)[marine sediments] 99 82 ES0502b1 100 clone Ucd1519(AM997592)[South-Atlantic Ocean sediment] EP2005-03c8(2) 100 clone B8A(FJ205243)[deep-sea hydrothermal region sediment] 99 E2005-01c16 clone 3FN(EF591886)[seawater] clone Hg91A1(EU236345)[sponge symbionts] 62 99 EP2005-03a30 EP2005-03c26 99 92 clone EPR4059-B2-Bc65(EU491565)[ocean crust] ES0502a23 55 clone MSB-3A12(DQ811897)[mangrove soil] Planctomycetes 100 ES0502a25 94 100 clone plankton_D05(FJ664799)[microbial fuel cells] ES0502b6 EP2005-03a35 100 clone EPR3970-MO1A-Bc78(EU491664)[ocean crust] 95 E2005-01a3 clone 086B35(EU734992)[Northern Bering Sea sediment] 100 E2005-01a30 clone MD2898-B17(EU386059)[South China Sea sediment] 87 EP2005-03b26 100 clone Ucb1551(AM997801)[South-Atlantic Ocean sediment] ES0502a11 ES0502c16 99 clone SC3-8(DQ289943)[permeable shelf sediment] EP2005-03c28 clone MD2.41(FJ403090)[coral] 0.01 EP2005-03c3

Fig. 8. Phylogenetic tree of Bacteroidetes, Chloroflexi, Candidate division TM6, Planctomycetes, and Verrucomicrobia from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. 582 Wang et al.

91 EP2005-03c12 Gemmatimonadetes 100 clone Urania-1B-32(AY627540)[Eastern Mediterranean sediment] WS0505c2 99 99 clone MD2902-B3 (EU048610)[Xisha Trough sediment] EP2005-03b33 84 76 clone M4-38(EU682499)[estuary sediment] WS0505b28 E2005-01b26 61 100 80 ES0502a16 58 clone EPR3970-MO1A-Bc8(EU491666)[ocean crust] ES0502b26 100 clone wb1_A18(AF317745)[Nullarbor caves] 86 E2005-01a15 EP2005-03b8(2)

98 Unclassified bacteria 88 clone Cm1-50(GQ246387)[North Yellow Sea sediments] E2005-01a17 90 clone EPR3970-MO1A-Bc45(EU491631)[ocean crust] 100 99 ES0502a1 clone Ucb1575(AM997865)[South-Atlantic Ocean sediment] 68 WS0505a31(3) E2005-01b5(2) 100 clone SPG12_60_70_B37(FJ746291)[marine sediment] clone S26-120(EU287420)[Pacific Arctic surface sediment] EP2005-03b24 100 clone B78-87(EU287051)[arctic surface sediment] 100 ES0502c8 clone EPR3967-O2-Bc30(EU491779)[ocean crust] 71 Clostridium paradoxum strain DSM 7308T(Z69934)

53 Desulfotomaculum kuznetsovii strain 17(AY036904) Firmicutes Thermoanaerobacter italicus strain DSM 9252(AJ250846) clone I7A(FJ205359)[deep-sea hydrothermal region sediment] ES0502c10 100 66 clone P0X3b1D04(EU491346)[ocean crust] WS0505c16 93 88 E2005-01a6 85 clone MD2902-B8(EU048615)[Xisha Trough sediment] 100 EP2005-03a1 clone P7X3b4E06(EU491069)[ocean crust] ES0502b12 100 clone Ucp1599(AM997666)[South-Atlantic Ocean sediment] E2005-01b20(2) 100 EP2005-03b25 99 clone HCM3MC91_5C_FL(EU374029)[Mediterranean Sea sediment] E2005-01b21(2) 100 clone P9X2b3H10(EU491158)[ocean crust] EP2005-03a31 clone Ucp15402(AM997675)[South-Atlantic Ocean sediment]

53 EP2005-03a2(4) Unclassified bacteria clone EPR4059-B2-Bc20(EU491531)[ocean crust] 100 ES0502a27 clone Ucc1579(AM997920)[South-Atlantic Ocean sediment] 81 E2005-01a4 clone Ksed4 (EU035874)[deep-sea coral reef sediment] EP2005-03b1 100 94 clone MD2896-B89(EU385703)[South China Sea sediment] 98 EP2005-03c37 100 clone Ulrdd 14(AM997480)[South-Atlantic Ocean sediment] 100 EP2005-03b14 clone B78-32(EU286996)[Pacific Arctic sediment] 86 ES0502c1 99 clone p36h18ok(FJ479350)[grass prairie] 100 ES0502c18 clone P9X2b3F04(EU491137)[ocean crust] 78 ES0502c19 100 clone SWB34(AB294345)[groundwater]

0.01

Fig. 9. Phylogenetic tree of Gemmatimonadetes, Firmicutes, and unclassified bacteria from the four libraries. Clones sequenced in this study are in bold, and numbers in parentheses indicate the total similar sequences (•2) in OTUs. The environments where relatives were obtained are shown in square brackets. Bootstrap values less than 50% are not shown. Scale represents a 1% difference in sequence. Bacterial diversity associated with polymetallic nodules 583G

Thermoanaerobacter italicus strain DSM 9252, Desulfoto- Metal cycling and nodule formation maculum kuznetsovii strain 17, and Clostridium paradoxum The CCFZ is a polymetallic nodule province where nodules strain DSM 7308T. Unlike the other clones in Gemmati- are abundant. Because of the high concentration of heavy monadetes, OTU ES0502b26 was associated with a clone from metals, such as iron (Fe), manganese (Mn), nickel (Ni), and Nullarbor caves, a nonaqueous environment, and formed a cobalt (Co), the CCFZ is a distinctive and important distant branch from other clones within Gemmatimonadetes. environment. Bacterial communities living in this area are Unclassified bacteria, comprising 28 clones that were unable predicted to be unique, with some bacteria involved in metal to be assigned into a taxonomic group, distributed in three cycling and nodule formation. In fact, nearly half of the libraries, except library WS0505. A total of 11 unclassified sequences recovered from the sampling stations were related bacteria were branched between Firmicutes and Gemmati- to uncultured representatives from the ocean crust, the Pacific monadetes, including a sister branch of Gemmatimonadetes nodule province and marine basalts. The ocean crust and (cluster 1) and a distantly branched OTU EP2005-03b24 marine basalts belong to the big oceanic lithosphere in which (cluster 2). The remaining 17 unclassified clones formed 4 the rock is very reactive and contains large amount of Fe and distinct branches, including cluster 3 to cluster 6, and Mn compared with seawater (Santelli et al., 2008). Thus, the belonged to different phylogenetic groups that were unable to corresponding phylotypes represented by these sequences may be identified. These unclassified clones may belong to new potentially be involved in metal cycling or in the formation of groups waiting to be identified and may be unique in the nodules or crust. However, an interesting result was that many studied environment. clones obtained in this study shared high similarities with uncultured relatives recovered from the sediment of three Discussion basins (Angola, Cape, and Guinea basins) of the South- Atlantic Ocean (Schauer et al., 2009). The geographic distance Comparison of the four sampling stations between the sampling stations in the tropical northeast Pacific The four sampling stations in this study were located in and the South-Atlantic Ocean is large, but clones obtained different areas of the CCFZ (Fig. 1 and Table 1). Station from both areas were highly overlapped, suggesting that some WS0505 belonged to the west part of COMRA’s contract area, similar environment factors (possibly high metal levels) may while stations ES0502 and E2005-01 belonged to the east part. exist. Station EP2005-03 was close to Mexico and outside COMRA’s Mn(II)-oxidizing bacteria have been found in diverse contract area. The abundance of polymetallic nodules was phylotypes, including low G+C Gram-positive bacteria (e.g., highest in station WS0505 and decreased in stations ES0502 Bacillus spp.), Alphaproteobacteria (e.g., Hyphomicrobium sp. and E2005-01. However, much fewer nodules could be found and Pedomicrobium sp.), Betaproteobacteria (e.g., Leptothrix around station EP2005-03. The Fe2O3 and MnO content in spp.), Gammaproteobacteria (e.g., Pseudomonas spp.), and sediment was highest in station WS0505 and lowest in station Bacteroidetes (e.g., Cytophaga sp.) (Cahyani et al., 2007; Wang EP2005-03, while it was similar in stations ES0502 and E2005- et al., 2009a). The Mn-reducing bacteria have been identified 01 (Table 1). In addition, other characteristics varied between in the genera Shewanella, Geobacter, Bacillus, Desulfovibrio, stations (Table 1). Different bacterial communities were and Desulfotomaculum, among others (Cahyani et al., 2007). assumed to be associated with the four different stations. Usually, Mn-cycling bacteria can also catalyze the redox Actually, bacterial diversity and community structure were reaction of iron or some other metals. Identified phylotypes dissimilar in the four libraries constructed from the four related to metal metabolism were detected in the present stations. In libraries E2005-01 and ES0502, the most dominant study. Clones related to Hyphomicrobium sp. Ellin112 and group was Gammaproteobacteria, followed by Alphaproteo- Pedomicrobium manganicum strain ACM 3038 were found bacteria, while the predominant groups were Alphaproteo- within Hyphomicrobiaceae of Alphaproteobacteria. Pedo- bacteria and Deltaproteobacteria in libraries EP2005-03 and microbium is a genus of budding hyphal bacteria that have the WS0505, respectively. It was considered that the dominant ability to oxidize Mn through either enzymatic mechanisms or bacterial groups were largely related to their corresponding iron oxidation (Cox and Sly, 1997; Larsen et al., 1999). P. living environment, which is consistent with the geographic manganicum is able to oxidize Mn by first adsorbing Mn to the relationship and characteristics of the four sampling stations. surface and subsequently catalyzing the oxidation with the In addition, the subordinate groups distributed in the four help of a multicopper oxidase (Sly and Arunpairojana, 1987; libraries in different patterns (Fig. 3). The bacterial diversity Larsen et al., 1999; Ridge et al., 2007). These bacteria are also varied between libraries. Rarefaction analysis revealed predicted to be involved in Mn cycling based on the that the most diverse library was ES0502, while the three physiology of their relatives. other libraries had lower diversity, according to the rarefac- In addition, Pseudomonas was detected in Gammaproteo- tion curves (Fig. 2). Previous studies have shown that almost bacteria, which contained 14 clones related to Pseudomonas all environments are dominated by only a few preponderant sp. NB1-h. Many strains of Pseudomonas isolated from diverse phylotypes but with a long tail of rare taxa (Fuhrman, 2009). environments have the ability to oxidize Mn(II), especially the Thus, a small library containing approximately 100 clones well-studied strains Pseudomonas putida MnB1 and GB-1 reveals the most abundant taxa but misses most of the rare (Francis and Tebo, 2001). However, Pseudomonas sp. NB1-h, taxa, except in samples with extremely low diversity (Fuhrman, isolated from Japan Trench sediment at a depth of 6292 m 2009). Because of the high diversity revealed by the rarefaction (Miki Yanagibayashi, 1999), has not been reported to oxidize analysis, many rare taxa may not have been detected in our Mn(II). Because Pseudomonas stains have diverse metabolic libraries. abilities, it is only hypothesized that these bacteria grouped 584 Wang et al. into Pseudomonas may play a role in Mn cycling. Nevertheless, subterraneus strain Red1 and Desulfuromonadales bacterium Pseudomonas strains can form biofilms easily, which is helpful Tc37, respectively (Fig. 6). D. infernum strain BalphaG is a in the formation and growth of nodules. thermophilic sulfate-reducing bacterium isolated from a Bacteria grouped into Desulfuromonadales within Delta- North Sea petroleum reservoir (Rees et al., 1995). G. proteobacteria were associated with the isolates G. subterraneus strain Red1 can reduce elemental sulfur as well subterraneus strain Red1 and Desulfuromonadales bacterium as Fe(III) and Mn(IV) (Greene et al., 2009). These sulfate- Tc37. G. subterraneus strain Red1 is an anaerobic Fe(III)- and reducing bacteria may cooperate with sulfur-oxidizing bacteria Mn(IV)-reducing bacterium and can receive energy from the in the ecosystem and play an indispensable role in sulfur reduction of Fe(III) and Mn(IV) (Greene et al., 2009). cycling. Bacteria belonging to Desulfuromonadales have also been detected in the bacterial communities inhabiting manganese Nitrogen cycling-related bacteria nodules in rice field subsoils (Cahyani et al., 2007), indicating Nitrification and denitrification, carried out by nitrifiers and that Desulfuromonadales are possibly involved in manganese denitrifiers respectively, are two important parts in nitrogen cycling. Cytophaga sp. has been reported to be involved in cycling, especially in marine environments. Nitrifiers are Mn(II) oxidation (Gregory and Staley, 1982; Cahyani et al., usually aerobic chemoautotrophs, such as Nitrosospira and 2007). Bacteria closely associated with Cytophaga sp. NB1-m Nitrosomonas, while denitrifiers are generally anaerobic may play a role in Mn(II) oxidation. heterotrophs (Ward, 1996). In this study, four clones associated with Nitrospina sp. 3005 and Nitrospina gracilis strain Nb-3 Sulfur cycling-related bacteria belonging to Nitrospinaceae were detected. N. gracilis is a Sulfur is a critical element in nature. Sulfuration and sulfate nitrite-oxidizing bacteria isolated from the marine environment reduction are two indispensable metabolic pathways in the (Watson and Waterbury, 1971). Thus, it is predicted that these cycling of sulfur. In this study, a cluster of clones belonging to bacteria are nitrite-oxidizing bacteria. Based on the chemical Ectothiorhodospiraceae was detected. Ectothiorhodospiraceae analysis of sediments from the sampling stations, the average is a family of purple sulfur bacteria, including phototrophic concentration of nitrate is approximately 40-45 PM and and chemoautotrophic bacteria, that can produce sulfur increases slightly with depth (data not shown), indicating that globules outside of their cells (Imhoff, 2006; Tourova et al., nitrification is dominant in these samples. However, other 2007). Twelve genera have been established in the nitrogen cycling-related bacteria are waiting to be discovered Ectothiorhodospiraceae family according to List of Prokaryotic by expanding the libraries. names with Standing in Nomenclature (LPSN). Ectothiorho- dospira, Ectothiorhodosinus, Halorhodospira, and Thiorho- Conclusion dospira are phototrophic genera, while Thioalkalispira and Thioalkalivibrio are chemolithotrophic sulfur-oxidizing genera Phylogenetic and diversity analyses of bacterial communities (Tourova et al., 2007). However, it is hard to conclude whether were performed for four stations in the CCFZ. The 313 total these bacteria are phototrophic or chemotrophic based merely clones sequenced from the 4 libraries, except for 28 on the phylogenetic analysis of 16S rRNA. We hypothesize unclassified bacteria, were divided into 14 phylogenetic groups. that these bacteria may be related to sulfur oxidation through The most dominant phylum was Proteobacteria, but the non-phototrophic metabolism because the deep ocean is predominant class within Proteobacteria was different in each considered to be dark. library. High bacterial diversity was revealed by the rarefaction Interestingly, a group of clones belonging to Rhodo- analysis. Because the CCFZ is abundant in polymetallic spirillaceae within Alphaproteobacteria were obtained. The nodules, we attempted to relate the bacterial communities Rhodospirillaceae family contains mainly purple non-sulfur with their habitats with the hope that special bacterial patterns bacteria, which produce energy by photosynthesis. However, would be discovered. The 16S rRNA gene libraries have some bacteria of Rhodospirillaceae, e.g., Rhodospirillum rubrum, revealed some bacteria potentially related to metals (e.g., Mn can grow either chemotrophically without light or phototro- and Fe) cycling, which is a very important process in the phically in light (Golecki and Oelze, 1980). The phototrophs formation and growth of polymetallic nodules. In addition, are intermingled with non-phototrophs in the phylogenetic bacteria possibly involved in sulfur and nitrogen cycling have tree (Kawasaki et al., 1993). Clones were associated with P. been detected. This study gives a primary view of bacterial litoralis and Skermanella as well as a distantly related communities living in the fields of deep-sea polymetallic magnetite-containing magnetic Vibrio strain MV1. These nodules and is the first report of a molecular ecological study bacteria are predicted to play a role in the oxidation of of four stations in the CCFZ. Some interesting discoveries of hydrogen sulfide at low concentrations. Likewise, because it is special bacteria, such as metal cycling-related bacteria and dark in the deep sea, these bacteria may oxidize sulfur unclassified novel bacteria, are worth further studies. More compounds chemotrophically. information about the bacterial communities living in the Sulfate-reducing bacteria (SRB) are a group of anaerobic studied area can be obtained through culture experiments as a bacteria that reduce sulfate or other oxidized sulfur compounds complement. to sulfide, including Desulfobacterales, Desulfovibrionales, and Syntrophobacterales. In the present study, clones belonging Acknowledgements to Syntrophobacterales and Desulfuromonadales were retrieved within Deltaproteobacteria that were distantly related to This work was supported by grants from China Ocean Mineral identified bacteria Desulfoacinum infernum strain BalphaG, G. Resources R & D Association (COMRA) Special Foundation Bacterial diversity associated with polymetallic nodules 585G

(DYXM-115-01-3-01, DYXM-115-01-3-02), the Ministry of Margolis, S.V. and R.G. Burns. 1976. Pacific deep-sea manganese Science and Technology of China (863 Program, 2007AA021305) nodules: Their distribution, composition, and origin. Annu. Rev. and the National Natural Science Foundation of China Earth Planet. Sci. 4, 229-263. (40806066). Miki Yanagibayashi, Y.N.L.L.C.K. 1999. Changes in the microbial community in Japan trench sediment from a depth of 6292 m during cultivation without decompression. FEMS Microbiol. Lett. References 170, 271-279. Rees, G.N., G.S. Grassia, A.J. Sheehy, P.P. Dwivedi, and B.K.C. Patel. Alexis, K., C. Jean-Claude, and C. Philippe. 2006. Geochemical and 1995. Desulfacinum infernum gen. nov., sp. nov., a thermophilic biological recovery of the disturbed seafloor in polymetallic sulfate-reducing bacterium from a petroleum reservoir. Int. J. Syst. nodule fields of the Clipperton-Clarion Fracture Zone (CCFZ) at Bacteriol. 45, 85-89. 5,000-m depth. Limnol. Oceanogr. 51, 2033-2041. Ridge, J.P., M. Lin, E.I. Larsen, M. Fegan, A.G. McEwan, and L.I. Sly. Barns, S.M., E.C. Cain, L. Sommerville, and C.R. Kuske. 2007. 2007. A multicopper oxidase is essential for manganese oxidation Acidobacteria phylum sequences in uranium-contaminated and laccase-like activity in Pedomicrobium sp. ACM 3067. Environ. subsurface sediments greatly expand the known diversity within Microbiol. 9, 944-953. the phylum. Appl. Environ. Microbiol. 73, 3113-3116. Santelli, C.M., B.N. Orcutt, E. Banning, W. Bach, C.L. Moyer, M.L. Cahyani, V.R., J. Murase, E. Ishibashi, S. Asakawa, and M. Kimura. Sogin, H. Staudigel, and K.J. Edwards. 2008. Abundance and 2007. Bacterial communities in manganese nodules in rice field diversity of microbial life in ocean crust. Nature 453, 653-657. subsoils: Estimation using PCR-DGGE and sequencing analyses. Schauer, R., C. Bienhold, A. Ramette, and J. Harder. 2009. Bacterial Soil Sci. Plant Nutr. 53, 575-584. diversity and biogeography in deep-sea surface sediments of the Cox, T.L. and L.I. Sly. 1997. Phylogenetic relationships and uncertain South Atlantic Ocean. ISME J. 4, 159-170. taxonomy of Pedomicrobium species. Int. J. Syst. Bacteriol. 47, 377- Schloss, P.D. and J. Handelsman. 2005. Introducing DOTUR, a 380. computer program for defining operational taxonomic units and Francis, C.A. and B.M. Tebo. 2001. cumA Multicopper oxidase genes estimating species richness. Appl. Environ. Microbiol. 71, 1501- from diverse Mn(II)-oxidizing and non-Mn(II)-oxidizing Pseudo- 1506. monas strains. Appl. Environ. Microbiol. 67, 4272-4278. Sly, L.I. and V. Arunpairojana. 1987. Isolation of manganese- Fuhrman, J.A. 2009. Microbial community structure and its functional oxidizing Pedomicrobium cultures from water by micromani- implications. Nature 459, 193-199. pulation. J. Microbiol. Methods 6, 177-182. Glasby, G., P. Stoffers, A. Sioulas, T. Thijssen, and G. Friedrich. 1982. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Manganese nodule formation in the Pacific Ocean: a general Molecular evolutionary genetics analysis (MEGA) software theory. Geo-Marine Lett. 2, 47-53. version 4.0. Mol. Biol. Evol. 24, 1596-1599. Golecki, J.R. and J. Oelze. 1980. Differences in the architecture of Thiel, H. 2001. Use and protection of the deep sea - an introduction. cytoplasmic and intracytoplasmic membranes of three chemotro- Deep-Sea Res. Part II-Top. Stud. Oceanogr. 48, 3427-3431. phically and phototrophically grown species of the Rhodo- Tourova, T.P., E.M. Spiridonova, I.A. Berg, N.V. Slobodova, E.S. spirillaceae. J. Bacteriol. 144, 781-788. Boulygina, and D.Y. Sorokin. 2007. Phylogeny and evolution of Greene, A.C., B.K.C. Patel, and S. Yacob. 2009. Geoalkalibacter the family Ectothiorhodospiraceae based on comparison of 16S subterraneus sp. nov., an anaerobic Fe(III)- and Mn(IV)-reducing rRNA, cbbL and nifH gene sequences. Int. J. Syst. Evol. Microbiol. bacterium from a petroleum reservoir, and emended descriptions 57, 2387-2398. of the family Desulfuromonadaceae and the genus Geoalkali- Wang, X.H. and W.E.G. Müller. 2009. Marine biominerals: bacter. Int. J. Syst. Evol. Microbiol. 59, 781-785. Perspectives and challenges for polymetallic nodules and crusts. Gregory, E. and J.T. Staley. 1982. Widespread distribution of ability Trends Biotechnol. 27, 375-383. to oxidize manganese among freshwater bacteria. Appl. Environ. Wang, X.H., U. Schloßmacher, M. Wiens, H. Schröder, and W. Microbiol. 44, 509-511. Müller. 2009a. Biogenic origin of polymetallic nodules from the Imhoff, J. 2006. The Family Ectothiorhodospiraceae, pp. 874-886. The clarion-clipperton zone in the Eastern Pacific Ocean: Electron Prokaryotes. microscopic and EDX evidence. Marine Biotechnol. 11, 99-108. Kawasaki, H., Y. Hoshino, and K. Yamasato. 1993. Phylogenetic Wang, X.H., H.C. Schröder, M. Wiens, U. Schloßmacher, and W.E.G. diversity of phototrophic purple non-sulfur bacteria in the Proteo- Müller. 2009b. Manganese/polymetallic nodules: Micro-structural bacteria Į group. FEMS Microbiol. Lett. 112, 61-66. characterization of exolithobiontic- and endolithobiontic microbial Krsek, M. and E.M.H. Wellington. 1999. Comparison of different biofilms by scanning electron microscopy. Micron. 40, 350-358. methods for the isolation and purification of total community Ward, B.B. 1996. Nitrification and denitrification: Probing the DNA from soil. J. Microbiol. Methods 39, 1-16. nitrogen cycle in aquatic environments. Microb. Ecol. 32, 247-261. Larsen, E.I., L.I. Sly, and A.G. McEwan. 1999. Manganese(II) Watson, S.W. and J.B. Waterbury. 1971. Characteristics of two marine adsorption and oxidation by whole cells and a membrane fraction nitrite oxidizing bacteria, Nitrospina gracilis nov. gen. nov. sp. and of Pedomicrobium sp. ACM 3067. Arch. Microbiol. 171, 257-264. Nitrococcus mobilis nov. gen. nov. sp. Arch. Microbiol. 77, 203-230. Liao, L., X.W. Xu, C.S. Wang, D.S. Zhang, and M. Wu. 2009. Webster, G., C.J. Newberry, J.C. Fry, and A.J. Weightman. 2003. Bacterial and archaeal communities in the surface sediment from Assessment of bacterial community structure in the deep sub- the northern slope of the South China Sea. J. Zhejiang University - seafloor biosphere by 16S rDNA-based techniques: a cautionary Science B. 10, 890-901. tale. J. Microbiol. Methods 55, 155-164. Maidak, B.L., J.R. Cole, T.G. Lilburn, C.T. Parker, P.R. Saxman, R.J. Xu, M.X., P. Wang, F.P. Wang, and X. Xiao. 2005. Microbial diversity Farris, G.M. Garrity, G.J. Olsen, T.M. Schmidt, and J.M. Tiedje. at a deep-sea station of the Pacific Nodule Province. Biodivers. 2001. The RDP-II (Ribosomal Database Project). Nucleic Acids Conserv. 14, 3363-3380. Res. 29, 173-174.