Home > Online First > Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Citation: Jinyu Yang, Yangyang Feng, Xiulan Chen, Binbin Xie, Yuzhong Zhang, Mei Shi, Xiying Zhang. Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-019-1391-9

doi: 10.1007/s13131-019-1391-9

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

1.  State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao 266237, China
2.  Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
3.  College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
4.  Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing Technology of Shandong Province/Key Laboratory of Novel Food Resources Processing, Ministry of Agriculture and Rural Affairs, Jinan 250100, China

Corresponding author: Xiying Zhang, zhangxiying@sdu.edu.cn

Received Date: 2018-12-07
Web Publishing Date: 2019-12-01

Fund Project: The AoShan Talents Cultivation Program supported by Qingdao National Laboratory for Marine Science and Technology under contract No. 2017ASTCP-OS14; the National Natural Science Foundation of China under contract Nos 31670063, 31670497 and 31870052; the Taishan Scholars Program of Shandong Province under contract No. 2009TS079; the Science and Technology Basic Resources Investigation Program of China under contract No. 2017FY100804.

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The N-terminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Key words: protease-producing bacteria , diversity , extracellular proteases , protease families , N-terminal amino acid sequencing , South China Sea


Arnosti C. 2011. Microbial extracellular enzymes and the marine carbon cycle. Annual Review of Marine Science, 3: 401–425.


Arnosti C, Bell C, Moorhead D L, et al. 2014. Extracellular enzymes in terrestrial, freshwater, and marine environments: perspectives on system variability and common research needs. Biogeochemistry, 117(1): 5–21.


Azam F, Malfatti F. 2007. Microbial structuring of marine ecosystems. Nature Reviews Microbiology, 5(10): 782–791.


Boetius A, Lochte K. 1994. Regulation of microbial enzymatic degradation of OM in deep-sea sediments. Marine Ecology Progress, 104: 299–307.


Bridoux M C, Neibauer J, Ingalls A E, et al. 2015. Suspended marine particulate proteins in coastal and oligotrophic waters. Journal of Marine Systems, 143: 39–48.


Chen Xiulan, Xie Binbin, Bian Fei, et al. 2009. Ecological function of myroilysin, a novel bacterial M12 metalloprotease with elastinolytic activity and a synergistic role in collagen hydrolysis, in biodegradation of deep-sea high-molecular-weight organic nitrogen. Applied and Environmental Microbiology, 75(7): 1838–1844.


Chen Xiulan, Xie Binbin, Lu Jingtao, et al. 2007. A novel type of subtilase from the psychrotolerant bacterium Pseudoalteromonas sp. SM9913: catalytic and structural properties of deseasin MCP-01. Microbiology, 153(7): 2116–2125.


Chen Xiulan, Zhang Yuzhong, Gao Peiji, et al. 2003. Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913. Marine Biology, 143(5): 989–993.


Chenna R, Sugawara H, Koike T, et al. 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research, 31(13): 3497–3500.


Fabiano M, Danovaro R. 1998. Enzymatic activity, bacterial distribution, and organic matter composition in sediments of the ross sea (Antarctica). Applied and Environmental Microbiology, 64(10): 3838–3845


Gao Xiang, Wang Jue, Yu Daqi, et al. 2010. Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family. Proceedings of the National Academy of Sciences of the United States of America, 107(41): 17569–17574.


Geng Ce, Nie Xiangtao, Tang Zhichao, et al. 2016. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins. Scientific Reports, 6: 25012.


He Hailun, Guo Jun, Chen Xiulan, et al. 2012. Structural and functional characterization of mature forms of metalloprotease E495 from Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. PLoS One, 7(4): e35442.


Herbert R A. 1999. Nitrogen cycling in coastal marine ecosystems. FEMS Microbiology Reviews, 23(5): 563–590.


Jørgensen B B, Boetius A. 2007. Feast and famine-microbial life in the deep-sea bed. Nature Reviews Microbiology, 5(10): 770–781.


Jung S Y, Jung Y T, Oh T K, et al. 2007. Photobacterium lutimaris sp. nov., isolated from a tidal flat sediment in Korea. International Journal of Systematic and Evolutionary Microbiology, 57(2): 332–336.


Kessler E, Safrin M, Olson J C, et al. 1993. Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. Journal of Biological Chemistry, 268(10): 7503–7508


Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2): 111–120.


Li Huijuan, Tang Bailu, Shao Xuan, et al. 2016. Characterization of a new S8 serine protease from marine sedimentary Photobacterium sp. A5-7 and the function of its protease-associated domain. Frontiers in Microbiology, 7: 2016.


Li Yan, Wu Chaoya, Zhou Mingyang, et al. 2017. Diversity of cultivable protease-producing bacteria in Laizhou Bay sediments, Bohai Sea, China. Frontiers in Microbiology, 8: 405.


Lloyd K G, Schreiber L, Petersen D G, et al. 2013. Predominant archaea in marine sediments degrade detrital proteins. Nature, 496(7444): 215–218.


Long R A, Azam F. 2001. Antagonistic interactions among marine pelagic bacteria. Applied and Environmental Mircobiology, 67(11): 4975–4983.


Matsuyama H, Sawazaki K, Minami H, et al. 2014. Pseudoalteromonas shioyasakiensis sp. nov., a marine polysaccharide-producing bacterium. International Journal of Systematic and Evolutionary Microbiology, 64(1): 101–106.


Moore E K, Harvey H R, Faux J F, et al. 2014. Electrophoretic extraction and proteomic characterization of proteins buried in marine sediments. Chromatography, 1(4): 176–193.


Nelson P N, Baldock J A. 2005. Estimating the molecular composition of a diverse range of natural organic materials from solid-state 13C NMR and elemental analyses. Biogeochemistry, 72(1): 1–34.


Olivera N L, Sequeiros C, Nievas M L. 2007. Diversity and enzyme properties of protease-producing bacteria isolated from sub-Antarctic sediments of Isla de Los Estados, Argentina. Extremophiles, 11(3): 517–526.


Patel A B, Fukami K, Nishijima T. 2001. Extracellular proteolytic activity in the surface sediment of a eutrophic inlet. Microbes and Environments, 16(1): 25–35.


Poremba K. 1995. Hydrolytic enzymatic activity in deep-sea sediments. FEMS Microbiology Ecology, 16(3): 213–221.


Qin Qilong, Zhang Xiying, Wang Xumin, et al. 2010. The complete genome of Zunongwangia profunda SM-A87 reveals its adaptation to the deep-sea environment and ecological role in sedimentary organic nitrogen degradation. BMC Genomics, 11: 247.


Ran Liyuan, Su Hainan, Zhao Guoyan, et al. 2013. Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family. Molecular Microbiology, 90(5): 997–1010.


Ran Liyuan, Su Hainan, Zhou Mingyang, et al. 2014. Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism. Journal of Biological Chemistry, 289(9): 6041–6053.


Rawlings N D, Barrett A J, Thomas P D, et al. 2018. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Research, 46(D1): D624–D632.


Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(Web server issue): W320–W324.


Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4): 406–425.


Schindler C A, Schuhardt V T. 1964. Lysostaphin: A new bacteriolytic agent for the Staphylococcus. Proceedings of the National Academy of Sciences of the United States of America, 51(3): 414–421.


Talbot V, Bianchi M. 1997. Bacterial proteolytic activity in sediments of the Subantarctic Indian Ocean sector. Deep Sea Research Part Ⅱ: Topical Studies in Oceanography, 44(5): 1069–1084.


Tamura K, Peterson D, Peterson N, et al. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10): 2731–2739.


Tsuboi S, Yamamura S, Imai A, et al. 2014. Linking temporal changes in bacterial community structures with the detection and phylogenetic analysis of neutral metalloprotease genes in the sediments of a hypereutrophic lake. Microbes and Environments, 29(3): 314–321.


Yan Bingqiang, Chen Xiulan, Hou Xiaoyan, et al. 2009. Molecular analysis of the gene encoding a cold-adapted halophilic subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913: cloning, expression, characterization and function analysis of the C-terminal PPC domains. Extremophiles, 13(4): 725–733.


Yang Xiangsheng, Chen Xinglin, Xu Xianzhong, et al. 2011. Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp. 547: enzyme characterization and gene cloning. Advances in Polar Science, 22(1): 49–54.


Yang Jian, Li Jie, Mai Zhimao, et al. 2013. Purification, characterization, and gene cloning of a cold-adapted thermolysin-like protease from Halobacillus sp. SCSIO 20089. Journal of Bioscience and Bioengineering, 115(6): 628–632.


Zhang Xiying, Han Xiaoxu, Chen Xiulan, et al. 2015. Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China. Frontiers in Microbiology, 6: 1021.


Zhao Huilin, Chen Xiulan, Xie Binbin, et al. 2012. Elastolytic mechanism of a novel M23 metalloprotease pseudoalterin from deep-sea Pseudoalteromonas sp. CF6-2: cleaving not only glycyl bonds in the hydrophobic regions but also peptide bonds in the hydrophilic regions involved in cross-linking. Journal of Biological Chemistry, 287(47): 39710–39720.


Zhao Guoyan, Chen Xiulan, Zhao Huilin, et al. 2008. Hydrolysis of insoluble collagen by deseasin MCP-01 from deep-sea Pseudoalteromonas sp. SM9913: collagenolytic characters, collagen-binding ability of C-terminal polycystic kidney disease domain, and implication for its novel role in deep-sea sedimentary particulate organic nitrogen degradation. Journal of Biological Chemistry, 283(52): 36100–36107.


Zhou Mingyang, Chen Xiulan, Zhao Huilin, et al. 2009. Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea. Microbial Ecology, 58(3): 582–590.


Zhou Mingyang, Wang Guanglong, Li Dan, et al. 2013. Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica. PLoS One, 8(11): e79668.


Zimmerman A E, Martiny A C, Allison S D. 2013. Microdiversity of extracellular enzyme genes among sequenced prokaryotic genomes. The ISME Journal, 7(6): 1187–1199.


Guihao LI, Lei SU, Qianqian ZHANG, Xiaoli ZHANG, Jun GONG. Molecular diversity and biogeography of benthic ciliates in the Bohai Sea and Yellow Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1236-y


Haibo LI, Wuchang ZHANG, Yuan ZHAO, Li ZHAO, Yi DONG, Chaofeng WANG, Chen LIANG, Tian XIAO. Tintinnid diversity in the tropical West Pacific Ocean. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1148-x


Chunguang WANG, Zhenzu XU, Donghui GUO, Jiaqi HUANG, Mao LIN. Taxonomic notes on Hydroidomedusae (Cnidaria) from the South China Sea V: Families Laodiceidae, Lovenellidae, Malagazziidae, and Mitrocomidae (Leptomedusae). ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1309-y


Junhui LIN, Yaqin HUANG, Ucu Yanu ARBI, Heshan LIN, Muhammad Husni AZKAB, Jianjun WANG, Xuebao HE, Jianfeng MOU, Kun LIU, Shuyi ZHANG. An ecological survey of the abundance and diversity of benthic macrofauna in Indonesian multispecific seagrass beds. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1181-9


Xuezheng LIN, Liang ZHANG, Yanguang LIU, Yang LI. Bacterial and archaeal community structure of pan-Arctic Ocean sediments revealed by pyrosequencing. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1030-2


Ying WANG, Chendong GE, Xinqing ZOU. Evidence of China’s sea boundary in the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1011-5


Jiajia Chen, Xuhua Cheng, Xiao Chen. Eddy generation mechanism in the eastern South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-019-1409-3


Lei GUO, Wenhuan ZHAN, Fan ZHANG, Jinchang ZHANG, Yantao YAO, Jian LI, Yingci FENG, Mei CHEN, Gong CHENG. The influence of earthquakes on Zhubi Reef in the Nansha Islands of the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1008-0


Ruiyan ZHANG, Xiaogu WANG, Yadong ZHOU, Bo LU, Chunsheng WANG. The first record of Porcellanaster ceruleus (Echinodermata: Porcellanasteridae) in the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1324-z


Jihai DONG, Yisen ZHONG. The spatiotemporal features of submesoscale processes in the northeastern South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1277-2


Qian LIU, Xiaohui XIE, Xiaodong SHANG, Guiying CHEN, Hong WANG. Modal structure and propagation of internal tides in the northeastern South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-019-1473-1


Yi Yu, Hao-Ran Zhang, Jiangbo Jin, Yuntao Wang. Trends of sea surface temperature and sea surface temperature fronts in the South China Sea during 2003–2017. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-019-1416-4


Jinglong YAO, Lei YANG, Yeqiang SHU, Lili ZENG, Rui SHI, Ju CHEN, Tingting ZU, Chuqun CHEN. Comparison of summer chlorophyll a concentration in the South China Sea and the Arabian Sea using remote sensing data. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1138-4


Feiyan DU, Lianggen WANG, Zhenzu XU, Jiaqi HUANG, Donghui GUO. Taxonomic notes on Anthomedusae (Cnidaria: Hydrozoa: Hydroidomedusa) from the south-central South China Sea, with a new genus and four new species. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-018-1310-5


Xuanliang JI, Guimei LIU, Shan GAO, Hui WANG, Miaoyin ZHANG. Comparison of air-sea CO2 flux and biological productivity in the South China Sea, East China Sea, and Yellow Sea: a three-dimensional physical-biogeochemical modeling study. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1098-8


Zhaoyun WANG, Fangguo ZHAI, Peiliang LI. A shift in the upper-ocean temperature trends in the South China Sea since the late 1990s. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-016-0947-1


Jichao WANG, Jie ZHANG, Jungang YANG, Wendi BAO, Guoli WU, Qifeng REN. An evaluation of input/dissipation terms in WAVEWATCH III using in situ and satellite significant wave height data in the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1038-7


Yanmin WANG, Shaowen LIU, Feifei HAO, Yunlong ZHAO, Chunyan HAO. Geothermal investigation of the thickness of gas hydrate stability zone in the north continental margin of the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1014-2


Jia SUN, Guihua WANG, Juncheng ZUO, Zheng LING, Dahai LIU. Role of surface warming in the northward shift of tropical cyclone tracks over the South China Sea in November. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1061-8


Shuzong HAN, Yongbin FAN, Yangyang DONG, Shuangquan WU. A study on the relationships between the wave height and the El Niño in the north area of the South China Sea. ACTA OCEANOLOGICA SINICA, doi: 10.1007/s13131-017-1059-2

  • PDF Downloads()
  • Abstract Views()
  • HTML Views()

Figures And Tables

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Jinyu Yang, Yangyang Feng, Xiulan Chen, Binbin Xie, Yuzhong Zhang, Mei Shi, Xiying Zhang