RESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Geobacter/metabolismo , Proteoma/metabolismo , Acetatos/metabolismo , Adaptação Fisiológica , Proteínas de Bactérias/genética , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Geobacter/crescimento & desenvolvimento , Heme/metabolismo , Viabilidade Microbiana , Fenômenos Fisiológicos da Nutrição , Oxidantes/metabolismo , Oxirredução , Proteoma/genética , Proteômica , Succinatos/metabolismoRESUMO
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.
Assuntos
Geobacter/genética , Técnicas de Cultura Celular por Lotes , Meios de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Compostos Férricos/metabolismo , Geobacter/crescimento & desenvolvimento , Geobacter/metabolismo , Mutação , FenótipoRESUMO
Methanogens, thought to be present on early Earth, have a high requirement for Ni, suggesting that Ni utilization could be a potential biosignature for methanogens if enhanced Ni extraction from surrounding minerals accompanies methanogenic growth. To test the potential for such Ni extraction from minerals by methanogens, Ni release from Ni-containing silicate glass was measured in Ni-free growth medium in the presence of the methanogen Methanothermobacter thermoautotrophicus (average pH â¼7.0) and observed to be higher than an abiotic control (average pH â¼6.8). However, batch dissolution experiments and a siderophore assay indicate that cell exudates such as siderophores, low molecular weight organic acids, or lysates accompanying cell death are not responsible for the observed increase in Ni release rate. In addition, scanning electron microscopy (SEM) shows little to no evidence of direct microbe-mineral interactions such as biofilms or pitting. Instead, comparison with abiotic experiments suggests that changes in pH due to CO2 uptake may be responsible for enhanced dissolution in the presence of metabolizing cells. These results document that methanogens may not preferentially extract Ni from surrounding minerals although they may indirectly affect mineral reaction rates that are pH sensitive. Thus identifiable Ni biosignatures may not exist in the rock record to document the presence of methanogens on early Earth or Mars.