Coexisting protist-bacterial community accelerates protein transformation in microcosm experiments

Abstract

Proteins constitute the major portion of labile substances in the marine environment and are an important source of organic matter supporting marine ecosystems. However, previous studies have revealed that specific bacterial membrane proteins are refractory in the oceans. We here show by kinetic analyses of protease degradation activity using inactivated Pseudomonas aeruginosa (Pa) cells as a proteinaceous substrate that bacterial proteases are insufficient to completely hydrolyze proteins, which may partially cause the protein accumulation in seawater. Protease activity was monitored simultaneously in 8 microcosms subjected to differing conditions. Some Pa proteins were retained for 30 days in the presence of bacteria without protists, whereas the Pa proteins were completely disappeared in the presence of both, indicating that these proteins were substantially incorporated into protist biomass. Our result suggests that protists play an important role in the transformation of bacterial proteins in seawater. Our experiments also imply that the functional/taxonomic diversity should be taken into account when considering decomposition activity in marine environments.

Extracellular proteases are released by ciliates in defined seawater microcosms

“…Mirowave-killed P. aeruginosa as ciliate food P. aeruginosa strain eco3 (Nonaka et al, 2010) was cultured in medium with phosphate-deficient artificial seawater according to the method of Thao et al (2014). The medium was artificial seawater as described in Yamada et al (2000) supplemented with 0.1 M sodium N-2-hydroxyethyl piperazine-N 0 -2-ethanesulfonate (HEPES) (pH 7.0), 7 mM (NH 4 ) 2 SO 4 , 20 mM sodium succinate (pH 7.0), 0.1% (v/v) trace ion mixture described in Hancock et al (1981), 0.2 mM sodium phosphate buffer (pH 7.0) and 1% proteose peptone No.…”

“…Live and dead bacterial cells are recognizable by green and red colors, respectively. Ciliate enumeration was conducted as described by Thao et al (2014). Ciliate cells were fixed with glutaraldehyde (final concentration, 1% [v/v]) and incubated at 4 C for at least 1 h. Fixed samples were double stained with FITC mixture (final concentration, 0.5% [v/v]) and DAPI (final concentration, 1 mg ml À1 ) for 10 min in the dark.…”

“…Protists are generally believed to contain digestive enzymes in the food vacuole (Muller and T€ or€ o, 1962;Nagata and Kirchman, 1992). However, our previous paper (Thao et al, 2014) indicated another possibility that the positively produced or leaked protist proteases are helpful for bacteria to use high molecular matter. Karner et al (1994) also reported the potential role of nanoflagellates in ectohydrolase production.…”

“…Various microbes in the community can certainly contribute to the bulk enzyme pool and previous research has shown that heterotrophic protists play fundamental and unanticipated roles in the marine food web (Weber et al, 2012) as bacterial grazers (Sherr and Sherr, 2002) and nutrient remineralizers (Nagata, 2000). Therefore, it is prerequisite that enzyme sources other than bacteria should also be investigated (Mohapatra and Fukami, 2004;Salerno and Stoecker, 2009;Thao et al, 2014). Our recent work suggests that protists could be an important source of proteases (Thao et al, 2014).…”

“…Therefore, it is prerequisite that enzyme sources other than bacteria should also be investigated (Mohapatra and Fukami, 2004;Salerno and Stoecker, 2009;Thao et al, 2014). Our recent work suggests that protists could be an important source of proteases (Thao et al, 2014). Further investigation is required to determine the community strategies e which type of extracellular enzymes are produced by which organism, what benefits from hydrolysis products and the extent to which various organisms collaborate or compete for substrates.…”