No description available.

Microbial iron respiration contributes significantly to the biogeochemical cycling of metals and may be one of the earliest respiratory processes to have evolved on early earth. Metal-respiring microbes also hold great potential for use in microbial fuel cells for the generation of "green" energy and for remediation of radionuclides in contaminated environments. Despite its significance in global metal cycling processes, the molecular mechanism of Fe(III) respiration has yet to be determined. Unlike many other terminal electron acceptors, Fe(III) is a solid at circumneutral pH and, therefore, cannot come into direct contact with the microbial inner membrane: the site of terminal electron transfer in gram-negative bacteria. It is postulated that metal-respiring organisms have developed alternate strategies for the reduction of solid iron. One such strategy involves the production of an Fe(III)-solublizing ligand by the metal-respiring bacteria which solubilizes the Fe(III) prior to respiration, rendering the metal more easily accessible to the Fe(III) reductase complex. In this study, the genes involved in the solubilization of Fe(III) by the gram-negative dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 were determined using random mutagenesis to generate mutations in the wild-type genome and high-throughput square-wave voltammetry to screen for the attenuation of Fe(III) production in the mutants. Two mutants unable to solubilize Fe(III) were identified and designated d29 and d64. After mutation complementation analysis, it was determined that the point mutations were both located in type II secretion genes: gspG and gspE respectively, indicating that the type II secretion system is required for Fe(III) solubilization prior to respiration. It was also hypothesized that the ligand produced for Fe(III) solubilization during dissimilatory Fe(III) respiration was a siderophore: a small Fe(III)-chelating molecule produced by the cells for the assimilation of Fe(III) for growth. A siderophore biosynthesis gene (SO3031) and a siderophore ferric reductase gene (SO3034) were deleted in frame and the resultant mutants screened to determine whether they were capable of Fe(III) solubilization and reduction during anaerobic Fe(III) respiration. Both mutants retained Fe(III) solubilization and reduction activity, indicating that the siderophore Fe(III) assimilatory system is distinct from the Fe(III) solubilization system utilized during Fe(III) respiration. The work presented here is significant in that it describes a rapid screening method for identifying Fe(III) solubilization mutants, reports on the involvement of the type II secretion system in Fe(III) solubilization during iron respiration, and finally demonstrates that a dissimilatory metal reducing bacteria synthesizes and secretes Fe(III)-chelating molecules which are distinct from Fe(III)-siderophores.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 69-76). Also available via the Internet.

Bacteria, while traditionally studied in liquid suspensions, are often found in nature as biofilms, aggregates of cells enclosed in self-produced matrices. Cells in biofilms have a fitness advantage over those that are free-living, as the biofilm lifestyle is correlated with increased resistance to various assaults, including antimicrobials, UV exposure, and dehydration. These biofilm-associated characteristics have important clinical implications, as biofilm-based bacterial infections are a major cause of morbidity in immunocompromised individuals. With this increased resiliency, however, comes a major challenge that arises during biofilm growth: the formation of resource gradients. My thesis work focused on one particular gradient, that of oxygen, which is established in biofilms formed by Pseudomonas aeruginosa. This bacterium has multiple mechanisms for coping with limited access to oxygen, including a highly-branched respiratory system for optimal oxygen scavenging and production and utilization of redox-active molecules called phenazines. The purpose of this thesis has been to investigate the different strategies used by P. aeruginosa to deal with the oxygen limitation precipitated by the biofilm lifestyle. In Chapter 1, I will provide the necessary background for understanding the principles of redox balancing, metabolism, respiration, biofilm physiology, and phenazine utilization in P. aeruginosa. The work described in Chapter 2 provides evidence for the formation of a novel terminal oxidase complex that plays a biofilm-specific role in P. aeruginosa growth. The results in this chapter also suggest that specific terminal oxidase complexes differ in the timing of their contributions to biofilm growth and implicate the novel complex in mediating reduction of phenazines in biofilms. Chapter 3 expands upon the principle of metabolic versatility exemplified by the results discussed in Chapter 2. The research presented in this chapter looks at how varying the source of electrons that feed into the respiratory chain influences downstream electron transfer steps, including terminal oxidase activities and phenazine production and utilization. The data presented in Chapters 2 and 3 add to the growing body of evidence that bacterial growth in liquid culture is distinct from that in biofilms and underscores the need for more biofilm-based research that can inform treatment strategies for P. aeruginosa infections. The results described in Chapter 4 take an even broader look at the strategies used by P. aeruginosa to sustain efficient metabolism under conditions of potential stress. An important node of central metabolism is pyruvate, which can be transformed in a number of ways. In this chapter, I will consider two pathways of pyruvate metabolism: fermentation to lactate and carboxylation to oxaloacetate. I will present data indicating that a previously-uncharacterized lactate dehydrogenase contributes to P. aeruginosa growth under specific growth conditions and that pyruvate carboxylation contributes to optimal progress through central metabolic pathways. I will also describe experiments that characterize the contributions of another carboxylase, previously thought to function as the pyruvate carboxylase, to P. aeruginosa’s ability to grow on selected nutrient sources. Finally, I will discuss how redox state informs biofilm formation in a phylogenetically distinct bacterium, Bacillus subtilis, highlighting the universality of redox reactions in driving metabolic processes. In sum, the research presented in this thesis broadens our understanding of the immense respiratory and metabolic flexibility of P. aeruginosa and serves as an important reminder of the discrete factors that govern liquid culture and biofilm growth.

No description available.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 60-68). Also available via the Internet.

Gluconobacter oxydans is characterized by extreme acid tolerance and the ability to carry out rapid, single-step polyol oxidation catalyzed by membrane bound ehydrogenases. Experiments were designed to determine whether acid tolerance is associated with rapid polyol oxidation in this organism. Washed cells were exposed to 0.1 M or 0.5 !vt NaCI at pH 3.20; subsequent alkalinization of the suspending solution suggested a NaCl-dependent flow of protons (H+) into the cells. Cells were then exposed to NaCI at pH 3.20 followed by the addition of glycerol to determine whether polyol oxidation resulted in H + explusion from the cells. Following glycerol addition, immediate acidification of the suspending solution occurred. To verify that H + effiux was a result of respiration, experiments were conducted using sodium azide and 2,4-dinitrophenol; both compounds prevented the acidification that otherwise occurred following glycerol addition. Because glycerol oxidation reversed the NaCl-induced flow of H + into the cell, it appeared that respiration might function to protect acid-labile cell interiors. Cells exposed to NaCl at pH 3.20 in the presence of glycerol maintained cellular viability while loss of viability occurred in the absence of glyceroL To verify the effect of H+ extrusion on pH homeostasis, radioactively labeled organic-acid probes were used to determine intracellular pH in respiring and nonrespiring cells in the presence of 0.1 M NaCI at pH 3.20. No differences in cytoplasmic pH values between respiring and nonrespiring cells were detected. However, because substantial evidence exists for the role of respiration dependent H + extrusion in the acid tolerance of G. oxydans, use of an alternate method for measurement of internal pH, such as 31 P nuclear magnetic resonance spectroscopy, is suggested. / Master of Science

Metal-respiring bacteria occupy a central position in a variety of environmentally important processes including the biogeochemical cycling of metals and carbon, biocorrosion of steel surfaces, bioremediation of radionuclide-contaminated aquifers, and electricity generation in microbial fuel cells. Metal-respiring bacteria are presented, however, with a unique physiological challenge: they are required to respire anaerobically on electron acceptors (e.g., Fe(III) oxides, elemental sulfur) that are highly insoluble at circumneutral pH and unable to enter the cell and contact inner membrane-localized respiratory systems. To overcome these physiological problems, metal-respiring bacteria are postulated to employ a variety of novel respiratory strategies not found in other bacteria, including 1) direct enzymatic reduction at the cell surface, 2) electron shuttling between the cell and metal surfaces, and 3) metal solubilization by bacterially-produced organic ligands followed by respiration of the soluble organic-metal complexes. This work highlights my latest findings on the genetic and enzymatic mechanism of metal respiration by Shewanella oneidensis, a facultative anaerobe ubiquitous to redox-stratified natural waters and sediments.

No description available.

The relationship between temperature and soil respiration has been well explored although uncertainties remain. This thesis examined the relationship between temperature and rates of heterotrophic respiration in soils from three adjacent sub-alpine Australian vegetation types; woodland, shrubland and grassland. Temperature sensitivity of soil (Q10) has recently been a hotly debate topic, one side concluding that decomposition of recalcitrant, less labile components of soil organic matter are insensitive to temperature. Whilst others argue that there is no difference in the temperature sensitivities of labile and recalcitrant carbon pools. Robust modeling of rates of soil respiration requires characterization of the temperature response of both labile and recalcitrant pools. Laboratory incubation provides a means of characterizing the temperature response of rates of respiration whilst reducing the confounding effects encountered in the field, such as seasonal fluctuations in temperature, moisture and substrate supply. I used a novel system that allowed laboratory measurement of gas exchange in soils over a range of temperatures under controlled conditions. Measurements included CO2 efflux and O2 uptake over a range of temperatures from 5 to 40oC, characterization of temperature response and sensitivity, and respiratory quotients. Rates of heterotrophic respiration fitted both exponential and Arrhenius functions and temperature sensitivity varied and depended on the model used, vegetation type and depth in the soil profile. Long-term incubation indicated both labile and resistant pools of carbon had similar temperature sensitivities. Respiratory quotients provided a strongly predictive measure of the potential rate of decomposition of soil C, independent of the temperature response of respiration, providing a tool that may be used alongside derived parameters to help understand shifts in microbial use of C substrates. Vegetation type influenced soil chemical properties and rates of heterotrophic respiration. Rates of respiration correlated well with concentrations of carbon and nitrogen as has been previously observed, unlike previous studies however a positive correlation was observed between indices of plant available phosphorus and respiration. The soils examined were from three adjacent vegetation types formed on common geology, I concluded that vegetation type had a significant influence on soil, in contrast to the commonly held view by ecologists that soil type drives patterns in vegetation. Climatic effects such as longer, dryer hotter summer, reduced snow cover and increased incidence of extreme weather events such as frosts and bushfire are likely to drive patterns in vegetation in this region and therefore have a significant impact on carbon cycling in Sub-alpine Australian soils.