The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanellaoneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research

Investigation of Yersinia pestis Laboratory Adaptation through a Combined Genomics and Proteomics Approach

The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement