Metaproteomics of marine viral concentrates reveals key viral populations and abundant periplasmic proteins in the oligotrophic deep chlorophyll maximum of the South China Sea

Abstract

Viral concentrates (VCs), containing bioinformative DNA and proteins, have been used to study viral diversity, viral metagenomics and virus-host interactions in natural ecosystems. Besides viruses, VCs also contain many noncellular biological components including diverse functional proteins. Here, we used a shotgun proteomic approach to characterize the proteins of VCs collected from the oligotrophic deep chlorophyll maximum (DCM) of the South China Sea. Proteins of viruses infecting picophytoplankton, that is, cyanobacteria and prasinophytes, and heterotrophic bacterioplankton, such as SAR11 and SAR116, dominated the viral proteome. Almost no proteins from RNA viruses or known gene transfer agents were detected, suggesting that they were not abundant at the sampling site. Remarkably, nonviral proteins made up about two thirds of VC proteins, including overwhelmingly abundant periplasmic transporters for nutrient acquisition and proteins for diverse cellular processes, that is, translation, energy metabolism and one carbon metabolism. Interestingly, three 56 kDa selenium-binding proteins putatively involved in peroxide reduction from gammaproteobacteria were abundant in the VCs, suggesting active removal of peroxide compounds at DCM. Our study demonstrated that metaproteomics provides a valuable avenue to explore the diversity and structure of the viral community and also the pivotal biological functions affiliated with microbes in the natural environment.

Illuminating microbial metabolic activities in the dark deep ocean with metaproteomics

“…As in our previous studies (25, 27, 28), the DS samples without purification for virions allowed us to identify exoproteins referring to the proteins present in the extracellular milieu. After two-step filtration, cell density (< 10 3 mL −1 ) of the DS fraction was below the detection limit of flow cytometry, indicating that the intact cells contributed little to the DS fraction.…”

“…The materials captured on a subsequent Durapore membrane filter (0.2 μm, Millipore) were used for the SP fraction (0.2 - 0.7 μm), while the filtrates were collected as the DS fraction. The additional concentration steps for the DS fraction was were as previously described (28). In brief, the filtrates were concentrated onboard with a Millipore Pellicon 2 tangential flow filtration system with a 10 kDa regenerated cellulose filter, and further concentrated and desalted in a Millipore 50 mL stirred ultrafiltration cell on ice.…”

“…The procedure for protein extraction of the three fractions was slightly modified from our previous studies (18, 27, 28, 56). Briefly, filters for LP or SP fractions were cut into chips and suspended in lysis buffer (urea, thiourea, Triton X-100, carrier ampholytes,3-[(3-Cholamidopropyl)dimethylammonium]-1-propanesulfonate, protease inhibitor cocktail, and dithiothretol (DTT)), broken-down with an ultrasonic shaker, sonicated on ice, and finally centrifuged at 10000×g for one hour after one-hour incubation.…”

“…All the supernatant was pooled together to be precipitated with ice-cold 20% trichloroacetic acid in acetone overnight. Protein extraction from the concentrated DS fraction was the same as in a previous study (28).…”

“…Proteins as the carrier and the functional executor of life can indicate information on metabolic activities and provide clues into the decomposition and degradation of organic matter (17). Recently, the emergence of metaproteomics has greatly broadened our knowledge of marine microbial activities by identifying proteins in the particulate fraction (18–24) and the dissolved fraction (25–28). However, these studies typically focus on a certain microbial fraction, resulting in partial snapshots of in situ microbial behavior.…”