Ecological functions of uncultured microorganisms in the cobaltrich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing

HUO Yingyi; CHENG Hong; Anton F. Post; WANG Chunsheng; JIANG Xiawei; PAN Jie; WU Min; XU Xuewei

doi:10.1007/s13131-015-0650-7

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Article Navigation > Acta Oceanologica Sinica > 2015 > 34(4): 92-113

HUO Yingyi, CHENG Hong, Anton F. Post, WANG Chunsheng, JIANG Xiawei, PAN Jie, WU Min, XU Xuewei. Ecological functions of uncultured microorganisms in the cobaltrich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing[J]. Acta Oceanologica Sinica, 2015, 34(4): 92-113. doi: 10.1007/s13131-015-0650-7

Citation:

HUO Yingyi, CHENG Hong, Anton F. Post, WANG Chunsheng, JIANG Xiawei, PAN Jie, WU Min, XU Xuewei. Ecological functions of uncultured microorganisms in the cobaltrich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing[J]. Acta Oceanologica Sinica, 2015, 34(4): 92-113. doi: 10.1007/s13131-015-0650-7

Citation:

HUO Yingyi, CHENG Hong, Anton F. Post, WANG Chunsheng, JIANG Xiawei, PAN Jie, WU Min, XU Xuewei. Ecological functions of uncultured microorganisms in the cobaltrich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing[J]. Acta Oceanologica Sinica, 2015, 34(4): 92-113. doi: 10.1007/s13131-015-0650-7

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Ecological functions of uncultured microorganisms in the cobaltrich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing

doi: 10.1007/s13131-015-0650-7

1.
Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012, China;State Key Laboratory of Satellite Ocean Environment Dynamics, Second Institute of Oceanography, Hangzhou 310012, China
2.
College of Life Sciences, Zhejiang University, Hangzhou 310058, China
3.
The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, Woods Hole, MA 02543, USA
4.
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China

Received Date: 2014-01-03
Rev Recd Date: 2014-07-05

Abstract

Abstract

Cobalt-rich ferromanganese is an important seafloor mineral and is abundantly present in the seamount crusts. Such crusts form potential hotspots for biogeochemical activity and microbial diversity, yet our understanding of their microbial communities is lacking. In this study, a cultivation-independent approach was used to recover genomic information and derive ecological functions of the microbes in a sediment sample collected from the cobalt-rich ferromanganese crust of a seamount region in the central Pacific. A total of 78 distinct clones were obtained by fosmid library screening with a 16S rRNA based PCR method. Proteobacteria and MGI Thaumarchaeota dominated the bacterial and archaeal 16S rRNA gene sequence results in the microbial community. Nine fosmid clones were sequenced and annotated. Numerous genes encoding proteins involved in metabolic functions and heavy metal resistance were identified, suggesting alternative metabolic pathways and stress responses that are essential for microbial survival in the cobalt-rich ferromanganese crust. In addition, genes that participate in the synthesis of organic acids and exoploymers were discovered. Reconstruction of the metabolic pathways revealed that the nitrogen cycle is an important biogeochemical process in the cobalt-rich ferromanganese crust. In addition, horizontal gene transfer (HGT) events have been observed, and most of them came from bacteria, with some occurring in archaea and plants. Clone W4-93a, belonging to MGI Thaumarchaeota, contained a region of gene synteny. Comparative analyses suggested that a high frequency of HGT events as well as genomic divergence play important roles in the microbial adaption to the deep-sea environment.
- seamount,
- cobalt-rich ferromanganese crust,
- metagenome,
- horizontal gene transfer

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References(24)

References

Adachi O, Ano Y, Toyama H, et al. 2007. Biooxidation with PQQ- and FAD-dependent dehydrogenases. In: Schmid R D, Urlacher V B, eds. Modern Biooxidation: Enzymes, Reactions and Applications. Hoboken, NJ: John Wiley & Sons, Inc, 41

Béjà O, Koonin E V, Aravind L, et al. 2002. Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces. Applied and Environmental Microbiology, 68(1): 335-345

Beman J M, Popp B N, Alford S E. 2012. Quantification of ammonia oxidation rates and ammonia-oxidizing archaea and bacteria at high resolution in the Gulf of California and eastern tropical North Pacific Ocean. Limnology and Oceanography, 57(3): 711-726

Blainey P C, Mosier A C, Potanina A, et al. 2011. Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis. PLoS One, 6(2): e16626

Candela T, Fouet A. 2006. Poly-gamma-glutamate in bacteria. Molecular Microbiology, 60(5): 1091-1098

Clark M R, Rowden A A, Schlacher T, et al. 2010. The ecology of seamounts: structure, function, and human impacts. Ann Rev Mar Sci, 2: 253-278

Craig J D, Andrews J E, Meylan M A. 1982. Ferromanganese deposits in the Hawaiian Archipelago. Marine Geology, 45(1-2): 127-157

Delaney M L. 1998. Phosphorus accumulation in marine sediments and the oceanic phosphorus cycle. Global Biogeochemical Cycles, 12(4): 563-572

Dell'Anno A, Danovaro R. 2005. Extracellular DNA plays a key role in deep-sea ecosystem functioning. Science, 309(5744): 2179

DeLong E F. 1992. Archaea in coastal marine environments. Proceedings of the National Academy of Sciences of the United States of America, 89(12): 5685-5689

Duffy E J. 2008. "Seamount". CenSeam: a global census of marine life on seamounts content partner and national oceanic and atmospheric administration content source. In: Cleveland C J, ed. Encycleopedia of Earth. Washington DC: Environmental Information Coalition, National Council for Science and the Environment

Ehrhardt C J, Haymon R M, Lamontagne M G, et al. 2007. Evidence for hydrothermal Archaea within the basaltic flanks of the East Pacific Rise. Environmental Microbiology, 9(4): 900-912

Emerson D, Moyer C L. 2010. Microbiology of seamounts: common patterns observed in community structure. Oceanography, 23: 148-163

Finkel S E. 2006. Long-term survival during stationary phase: evolution and the GASP phenotype. Nat Rev Microbiol, 4(2): 113-120

Finkel S E, Kolter R. 2001. DNA as a nutrient: novel role for bacterial competence gene homologs. Journal of Bacteriology, 183(21): 6288-6293

Fu Yazhou, Peng Jiantang, Qu Wenjun, et al. 2005. Os isotopic compositions of a cobalt-rich ferromanganese crust profile in Central Pacific. Chin Sci Bull, 50(18): 2106-2112

Gadd G M. 2007. Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycological Research, 111(Pt 1): 3-49

García-Martínez J, Martínez-Murcia A, Antón A I, et al. 1996. Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12. Journal of Bacteriology, 178(21): 6374-6377

Ghiorse W C. 1984. Biology of iron- and manganese-depositing bacteria. Annu Rev Microbiol, 38: 515-550

Gillan D C, Danis B. 2007. The archaebacterial communities in Antarctic bathypelagic sediments. Deep-Sea Research Part II: Topical Studies in Oceanography, 54(16-17): 1682-1690

Guibaud G, van Hullebusch E, Bordas F, et al. 2009. Sorption of Cd(II) and Pb(II) by exopolymeric substances (EPS) extracted from activated sludges and pure bacterial strains: Modeling of the metal/ligand ratio effect and role of the mineral fraction. Bioresource Technology, 100(12): 2959-2968

Heijs S K, Haese R R, van der Wielen P W, et al. 2007. Use of 16S rRNA gene based clone libraries to assess microbial communities potentially involved in anaerobic methane oxidation in a Mediterranean cold seep. Microbial Ecology, 53(3): 384-398

Hillier J K, Watts A B. 2007. Global distribution of seamounts from ship-track bathymetry data. Geophysical Research Letters, 34(13): L13304

Ito M, Tsunekawa M, Yamaguchi E, et al. 2008. Estimation of degree of liberation in a coarse crushed product of 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