(796h) Label-Free, Strain-Resolved, Shotgun Proteomics of a Defined Bacterial Co-Culture | AIChE

(796h) Label-Free, Strain-Resolved, Shotgun Proteomics of a Defined Bacterial Co-Culture


Reardon, K., Colorado State University
Park, S., Colorado State University

Systems biology approaches can provide new, detailed insights into the interactions among members of communities of microorganisms that are unavailable through conventional methods. An “-omics” approach provides metabolic information about the effects of interactions on different community members, thereby suggesting functional mechanisms to explain dynamics of the system as a whole. Recently, analysis of defined co-culture systems has been identified as a horizon for metabolic modeling and bioprocessing. Strain-resolved proteomics can track proteome changes of each species of a co-culture. To evaluate the complexity of molecular-level responses associated with growth in community, a quantitative, shotgun, label-free proteomics study was conducted on a defined bacterial co-culture consisting of two well-characterized soil species: Gram-negative Pseudomonas putida KT2440 and Gram-positive Bacillus atrophaeus ATCC 9372, the standard surrogate for the pathogen Bacillus anthracis. A total of 1151 proteins were identified across pure and co-culture samples. Differentially-expressed proteins were quantified by two methods: precursor ion intensity and spectral counts. Proteins were categorized according to Gene Ontology categories and KEGG pathways. Twenty-seven B. atrophaeus proteins and 50 P. putida proteins were significantly more abundant in the co-culture than in the respective pure culture (p<0.05; fold-change > 2), while 137 B. atrophaeus and 42 P.putida proteins were significantly less abundant in the co-culture, compared to the pure culture (p<0.05; fold-change < 2). For both species, the most common Gene Ontology biological process category for upregulated proteins was regulation of transcription. For B. atrophaeus, nearly 15% of the proteins upregulated in coculture were related to stress response. These proteins included GrpE (4. fold higher in co-culture compared to pure culture), penicillin-binding lipoprotein 3 (2 fold higher in co-culture) and glycosyltransferase (3.7 fold higher in co-culture), the latter two of which are a response to antibiotic or toxin. For P.putida, several proteins were related to toxin biosynthesis, including PPM/P mutase (2.2 fold higher in co-culture than in pure culture), 2-dehydro-3-deoxyphosphooctonate aldolase 2 (2. fold higher in co-culture), phenazine biosynthesis protein (6.5 fold higher than in co-culture), and GTP cyclohydrolase-2 (2.3 fold higher in co-culture). Futhermore, for each species several differentially-expressed proteins related to iron uptake. For B.atrophaeus, 2,3-dihydroxybenzoate-AMP ligase, an enzyme in the siderophore biosynthesis pathway, was 7-fold higher in the co-culture than in pure culture. Bacterioferritin was down-regulated in P.putida. Follow-up growth studies involving growth in culture supernatant and at different levels of iron availability were conducted to verify interactions suggested by the proteomics data. This study contributes to a growing literature that seeks to discover and characterize patterns of microbial interactions through powerful “-omics” analysis of co-culture systems of model species.