Survival during long-term starvation: global proteomics analysis of Geobacter sulfurreducens under prolonged electron-acceptor limitation.

The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under "feast or famine", in the laboratory most organisms are studied under "feast" conditions, whereas they typically encounter "famine" in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulfurreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (p < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation.

Growth advantage in stationary-phase (GASP) phenotype in long-term survival strains of Geobacter sulfurreducens.

Geobacter sulfurreducens exists in the subsurface and has been identified in sites contaminated with radioactive metals, consistent with its ability to reduce metals under anaerobic conditions. The natural state of organisms in the environment is one that lacks access to high concentrations of nutrients, namely electron donors and terminal electron acceptors (TEAs). Most studies have investigated G. sulfurreducens under high-nutrient conditions or have enriched for it in environmental systems via acetate amendments. We replicated the starvation state through long-term batch culture of G. sulfurreducens, where both electron donor and TEA were scarce. The growth curve revealed lag, log, stationary, death, and survival phases using acetate as electron donor and either fumarate or iron(III) citrate as TEA. In survival phase, G. sulfurreducens persisted at a constant cell count for as long as 23 months without replenishment of growth medium. Geobacter sulfurreducens demonstrated an ability to acquire a growth advantage in stationary-phase phenotype (GASP), with strains derived from subpopulations from death- or survival phase being able to out-compete mid-log-phase populations when co-cultured. The molecular basis for GASP was not because of any detectable mutation in the rpoS gene (GSU1525) nor because of a mutation in a putative homolog to Escherichia coli lrp, GSU3370.

Induction of SIV p27-specific multifunctional T cells in the gut following prime-boost immunization with Clostridium perfringens and adenovirus vaccines expressing SIV p27.

A vaccine-induced cellular immune response to simian immunodeficiency virus (SIV) in the gut mucosal tissue may prevent the establishment or severity of new SIV infection. An oral Clostridium perfringens expressing SIV p27 (Cp-p27) vaccine that delivers SIV p27 to the gut was evaluated for its ability to prime multifunctional cellular immunity in the gut mucosa. Gut Peyer's patches dendritic cells matured in response to in vitro exposure to Cp-p27 and stimulated production of p27-specific IFN-gamma. In mice, the oral vaccination with the Cp-p27 vaccine and systemic immunization with adenovirus expressing SIV p27 (Ad-p27) induced robust systemic and mucosal immune responses. Furthermore, the prime-boost regimen induced p27-specific multifunctional CD8+ T cells in the gut. These results indicate that priming gut tissue with Cp-p27 can enhance the gut mucosal cellular immune response generated via systemic immunization with Ad-p27.