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

Proteomic Analysis of Growth Phase-Dependent Expression of Legionella pneumophila Proteins Which Involves Regulation of Bacterial Virulence Traits

Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization–Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase