Abstract
Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance.
Highlights
The extremely high diversity of marine microorganisms was shown ever since the first large metagenomic shotgun analysis carried out on total DNA extracted from filtered seawater samples collected from the Sargasso Sea near Bermuda [1]
The acquired tandem mass spectra were assigned with the Mascot software using as database the complete list of annotated Coding Domain Sequences (CDS) from Phaeobacter strain DSM 17395
Whether the expected abundant 55 kDa protein was encoded on the genome but missed during the annotation stage was investigated by a new Mascot search using a six-frame translation of the whole nucleic acid sequence, as previously described [34]. This proteogenomic approach remained unsuccessful with no considerable findings (Data not shown)
Summary
The extremely high diversity of marine microorganisms was shown ever since the first large metagenomic shotgun analysis carried out on total DNA extracted from filtered seawater samples collected from the Sargasso Sea near Bermuda [1]. Heterotrophic bacterioplankton communities in near-surface marine pelagic environments mostly comprise Alphaproteobacteria and Gammaproteobacteria, as well as Sphingobacteria and Flavobacteria (both belonging to the Bacteroidetes superphylum). As shown recently with environmental metagenomic data, prominent components belong to the SAR11, Roseobacter and SAR86 clades together with the OM60/ NOR5 cluster [2]. The Roseobacter lineage is a phylogenetically coherent group of Alphaproteobacteria comprising up to 20% of marine microbial communities, especially in coastal and polar waters [3]. The Roseobacter clade is a physiologically heterogeneous group showing a high variety of lithoheterotrophic strategies. Despite a free-living lifestyle, Roseobacter strains are found as planktonic or larger pluricellular symbionts [4]
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