Comparative proteogenomics of twelve Roseobacter exoproteomes reveals different adaptive strategies among these marine bacteria

Abstract

Roseobacters are generalist bacteria abundantly found in the oceans. Because little is known on how marine microorganisms interact in association or competition, we focused our attention on the microbial exoproteome, a key component in their interaction with extracellular milieu. Here we present a comparative analysis of the theoretically encoded exoproteome of twelve members of the Roseobacter group validated by extensive comparative proteogenomics. In silico analysis revealed that 30% of the encoded proteome of these microorganisms could be exported. The ratio of the different protein categories varied in accordance to the ecological distinctness of each strain, a trait reinforced by quantitative proteomics data. Despite the interspecies variations found, the most abundantly detected proteins by shotgun proteomics were from transporter, adhesion, motility, and toxin-like protein categories, defining four different plausible adaptive strategies within the Roseobacter group. In some strains the toxin-secretion strategy was over-represented with repeats-in-toxin-like proteins. Our results show that exoproteomes strongly depend on bacterial trophic strategy and can slightly change because of culture conditions. Simulated natural conditions and the effect of the indigenous microbial community on the exoproteome of Ruegeria pomeroyi DSS-3 were also assayed. Interestingly, we observed a significant depletion of the toxin-like proteins usually secreted by R. pomeroyi DSS-3 when grown in presence of a natural community sampled from a Mediterranean Sea port. The significance of this specific fraction of the exoproteome is discussed.

Fig. 1.

Multidomain structure of representative RTX polypeptides from 12 Roseobacter clade isolates. Boxes represent structural domains predicted by the CDD algorithm (NCBI) after PSI-BLAST analysis. An example of each RTX-toxin detected in each of the 12 bacteria analyzed in this study is represented. Stand-alone RTX polypeptides with only the typical serralysin peptidase domain are not indicated although frequently found. Numbers on the schematic structure represent amino acid lengths. The NCBI references of each protein are indicated as well as the corresponding bacterial strains.

Fig. 2.

SDS-PAGE analysis of exoproteomes obtained from Roseobacter clade strains. Exoproteins were resolved by a long migration on a 10% SDS-PAGE gels and stained with SimplyBlue SafeStain (Invitrogen). SeeBlue Plus2 marker from Invitrogen loaded in the first lane was used for molecular weight determination. A, shows the exoproteomes of 11 Roseobacter clade strains grown in MB until mid exponential phase. B, shows the exoproteomes of R. pomeroyi DSS-3 cells grown under the different conditions indicated at the top of each lane. The band containing PaxA is indicated with an arrow.

Fig. 3.

Relative abundance of MS/MS detected polypeptides in the different Roseobacter clade exoproteomes in terms of functional categories. The percentages of the different categories are graphed by stacked bars. Relative numbers of the different polypeptides, spectral counts and protein abundance normalized by the corresponding molecular weight are labeled for each strain CDS, SC, and NSAF, respectively. Location and origin of the initial sampling for the bacterium isolation are indicated (see www.roseobase.org for additional details). Abbreviations used are: Med. sea for Mediterranean sea, Dinofl. for Dinoflagellate, and 10m for isolations done from 10-meter deep seawater. Maximal doubling times (D.T.) established in marine broth in this study are indicated in hours. Trophic strategies assigned to each Roseobacter clade isolate are indicated by A, B, C, and D indicating a large production of nutrient transporters, mobility proteins, adhesion-like proteins, and toxin secretion, respectively.

Fig. 4.

Degree of conservation among Roseobacters of the 14 RTX proteins from R. pomeroyi and their genomic context. Polypeptides detected by MS/MS in the present study are indicated in red. Their relative abundance among exoproteins established by MS/MS spectral count is indicated for the five culture conditions. EXP, STA, LB, MM, and SW stand for MB exponential phase, MB stationary phase, 1:1 mixed MB and LB broths, MMM media and autoclaved seawater supplemented with succinate, respectively. The logarithmic E-values calculated for the best BLAST match in each Roseobacter strain (or second best in the case of R. pomeroyi) is indicated to show the conservation of these proteins among Roseobacters. Highest values are highlighted in bold. The 5-Kbp genomic region comprising the R. pomeroyi RTX-proteins are represented. Numbers in RTX-like proteins are the length of cut-out sequences in kbp necessary for representation of large genes. Coding genome strands are indicated as follows: Crick strand (+) and Watson strand (−). Colors indicated the functional categories of polypeptides encoded by the neighboring genes.