Proteomic analysis of carbohydrate catabolism and regulation in the marine bacteriumRhodopirellula baltica

Abstract

The marine bacterium Rhodopirellula baltica, a member of the phylum Planctomycetes, has distinct morphological properties and contributes to remineralization of biomass in the natural environment. On the basis of its recently determined complete genome we investigated its proteome by 2-DE and established a reference 2-DE gel for the soluble protein fraction. Approximately 1000 protein spots were excised from a colloidal Coomassie-stained gel (pH 4-7), analyzed by MALDI-MS and identified by PMF. The non-redundant data set contained 626 distinct protein spots, corresponding to 558 different genes. The identified proteins were classified into role categories according to their predicted functions. The experimentally determined and the theoretically predicted proteomes were compared. Proteins, which were most abundant in 2-DE gels and the coding genes of which were also predicted to be highly expressed, could be linked mainly to housekeeping functions in glycolysis, tricarboxic acid cycle, amino acid biosynthesis, protein quality control and translation. Absence of predictable signal peptides indicated a localization of these proteins in the intracellular compartment, the pirellulosome. Among the identified proteins, 146 contained a predicted signal peptide suggesting their translocation. Some proteins were detected in more than one spot on the gel, indicating post-translational modification. In addition to identifying proteins present in the published sequence database for R. baltica, an alternative approach was used, in which the mass spectrometric data was searched against a maximal ORF set, allowing the identification of four previously unpredicted ORFs. The 2-DE reference map presented here will serve as framework for further experiments to study differential gene expression of R. baltica in response to external stimuli or cellular development and compartmentalization.

Two-dimensional gel electrophoresis in bacterial proteomics

“…2D-DIGE is capable of quantifying small differences in protein levels, making it a popular approach for large-scale expression profiling in a range of biological systems. In the field of bacterial proteomics, 2D-DIGE has become a standard comparative proteomic approach for the study of gene regulation, stress responses, virulence mechanisms as well as environmental adaptation in both Gram-negative and Gram-positive species (Gade et al, 2005;Al Dahouk et al, 2008;Alteri et al, 2009;Franco et al, 2009). Investigators have previously compared directly the use of 2D-DIGE and colorimetric protein stains to quantify changes in protein expression patterns in E. coli cells treated with benzoic acid (Yan et al, 2002;Han and Lee, 2006).…”

Proteomics of marine bacteria

“…Varying levels of light intensity iTRAQ and LC-M/MS [43] Heterotrophy/photoautotrophy 2-D PAGE, MALDI-MS and LC-MS/MS [50] Nutrient availability/different C-and energy sources [68,74,75] Growth phase/cell cycle…”

“…It undergoes a cell cycle, differentiating between motile swarmer cells and sessile cell aggregates [66]. R. baltica's interesting physiological potential has been subject to several proteomic investigations during the previous years ([ [67][68][69]133]. A first 2-D reference map of cytosolic R. baltica proteins in the pH range of 4-7 was established by PMF in 2005, including 558 distinct proteins [67].…”

“…Using a comparative proteomic approach that investigated R. baltica's carbon catabolism, several so-called stimulons, i.e. distinct groups of carbon degradation-related proteins could be identified in cells grown with ribose, N-acetylglucosamine, xylose, maltose, lactose, melibiose and raffinose, respectively [68]. Interestingly, the authors found that coordinately regulated R. baltica proteins are not necessarily coded within the same genomic regions (as are for example all proteins involved in the catabolism of ribose).…”

“…Interestingly, the authors found that coordinately regulated R. baltica proteins are not necessarily coded within the same genomic regions (as are for example all proteins involved in the catabolism of ribose). Some functionally related proteins, like the ones involved in xylose degradation, the abundance of which increased in the presence of xylose, are not organized in clusters [68]. In an analogous study, the same authors investigated growth phase-dependent variations in the R. baltica protein pattern using the differential in gel electrophoresis (DIGE) technology [69].…”