Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 126-130). Also available on the World Wide Web.

<p>Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, <i>Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria</i>). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera <i>Pantoea</i> were found to inhibit growth of <i>E. coli</i> O157:H7 <i>in vitro</i>. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface.</p> / Master of Science

Celiac disease is an autoimmune enteropathy resulting from the ingestion of gluten and gluten-like proteins from wheat, barley and rye in afflicted individuals. Indigestible gluten-derived peptides rich in proline residues are known to be responsible for eliciting the inappropriate immune response characteristic of the disease. In this investigation, surface level expression of prolyl peptidase activity by genetically engineered probiotic lactobacilli was postulated to be a possible treatment for this disease. Plasmid-based reporter vectors were constructed utilizing a novel, homology-based cloning technique to assess the expression and localization signals from the S-layer protein gene of Lactobacillus acidophilus. These plasmids were mutated during construction due to toxicity associated with the cloned cassettes. The toxicity of the Slayer secretion and/or anchoring domains in E. coli was confirmed by cloning the fused components into an inducible expression system. When the prolyl peptidase, Xaa-Pro, from L. reuteri was incorporated into the S-layer expression cassette, the full-length protein was efficiently expressed but was not active, likely due to protein aggregation and inclusion body formation. Future research directions are discussed and a modified experimental design strategy is presented. This work provides a foundation for continued investigation into the feasibility of utilizing genetically engineered lactobacilli as a potential treatment strategy for celiac disease.

Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, <i>Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria</i>). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera <i>Pantoea</i> were found to inhibit growth of <i>E. coli</i> O157:H7 <i>in vitro</i>. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface. / Master of Science in Life Sciences / Master of Science

abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Washington State University, May 2009. / Title from PDF title page (viewed on June 8, 2009). "School of Electrical Engineering and Computer Science." Includes bibliographical references.

Yeung Heung-fun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1987. / Includes bibliographical references.

by Yip Chi Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 70-87). / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iv / TABLE OF CONTENTS --- p.v / LIST OF TABLES --- p.ix / LIST OF FIGURES --- p.xiii / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.3 / Chapter I. --- Mycobacterial Infections --- p.3 / Chapter A. --- Mycobacterium tuberculosis --- p.3 / Chapter B. --- Atypical mycobacteria --- p.3 / Chapter II. --- Identification of Mycobacteria --- p.5 / Chapter A. --- Conventional methods --- p.6 / Chapter 1. --- Mycobacterium tuberculosis --- p.6 / Chapter 2. --- Atypical mycobacteria --- p.7 / Chapter B. --- Rapid identification methods --- p.8 / Chapter 1. --- Identification by fatty acid analysis --- p.8 / Chapter 2. --- Identification by mycolic acid analysis --- p.9 / Chapter III. --- In vitro Susceptibility Testing of Mycobacteria --- p.11 / Chapter A. --- Mycobacterium tuberculosis --- p.11 / Chapter 1. --- Principle --- p.12 / Chapter 2. --- Methods of susceptibility testing --- p.13 / Chapter a. --- The absolute concentration method --- p.13 / Chapter b. --- The resistance ratio method --- p.15 / Chapter c. --- The 1% proportion method --- p.16 / Chapter d. --- Radiometric method --- p.18 / Chapter e. --- Other methods --- p.19 / Chapter B. --- Atypical mycobacteria --- p.20 / Chapter IV. --- Plasmid Analysis in Mycobacteria --- p.22 / Chapter A. --- Discovery of plasmids in mycobacteria --- p.22 / Chapter B. --- Methodologies in the studies of mycobacterial plasmids --- p.23 / Chapter C. --- Possible roles of plasmid in epidemiology of mycobacteria --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter I. --- Bacterial Strains and Strain Maintenance --- p.26 / Chapter A. --- Strains collection --- p.26 / Chapter B. --- Strains maintenance --- p.26 / Chapter II. --- Culture Media and Culture Conditions --- p.26 / Chapter III. --- Identification of Mycobacteria --- p.26 / Chapter A. --- Conventional methods --- p.26 / Chapter B. --- Fatty acid profile analysis --- p.27 / Chapter 1. --- Bacterial isolates --- p.27 / Chapter 2. --- Standards and reagents --- p.27 / Chapter 3. --- Preparation of methyl ester for GC/GC-MS --- p.28 / Chapter 4. --- Instrumentation --- p.28 / Chapter a. --- Gas chromatography-mass spectrometry (GC-MS) --- p.28 / Chapter b. --- Gas liquid chromatography (GLC) --- p.28 / Chapter 5. --- Fatty acid profile analysis --- p.29 / Chapter a. --- Calibration --- p.30 / Chapter b. --- Identification of mycobacterial fatty acids --- p.30 / Chapter c. --- Construction of mycobacterial fatty acid profiles --- p.30 / Chapter 6. --- Discriminant analysis --- p.31 / Chapter IV. --- In Vitro Drug Susceptibility Test --- p.31 / Chapter A. --- Test strains --- p.31 / Chapter B. --- Preparation of drug-containing media --- p.32 / Chapter C. --- Minmum inhibition concentration (MIC) determination --- p.32 / Chapter V. --- Heavy Metal Tolerance Test --- p.33 / Chapter A. --- Bacterial strains --- p.33 / Chapter B. --- Reagent and media preparation --- p.34 / Chapter 1. --- Heavy metal stock solution preparation --- p.34 / Chapter 2. --- Media preparation --- p.34 / Chapter C. --- Minimum inhibition concentration (MIC) determination --- p.34 / Chapter VI. --- Plasmid Analysis of Mycobacteria --- p.35 / Chapter A. --- Bacterial strains --- p.35 / Chapter B. --- Extraction procedures --- p.35 / Chapter 1. --- Modified Kado & Liu method --- p.35 / Chapter 2. --- French press procedure --- p.36 / Chapter 3. --- Spheroplasts preparation procedure --- p.37 / Chapter C. --- Electrophoresis procedure --- p.37 / Chapter D. --- Statistical analysis for correlation between plasmid and drug resistance or heavy metal tolerance --- p.38 / RESULTS --- p.39 / Chapter I. --- Identification of Mycobacteria --- p.39 / Chapter A. --- General characteristics of the chromatographic profile --- p.39 / Chapter B. --- Discriminant analysis --- p.40 / Chapter 1. --- Gas chromatography-mass spectrometry (GC-MS) --- p.40 / Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.40 / Chapter b. --- Rapidly growing mycobacter-ia --- p.41 / Chapter c. --- Pigmented mycobacteria --- p.41 / Chapter 2. --- Gas liquid chromatography (GLC) --- p.41 / Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.42 / Chapter b. --- Rapidly growing mycobacteria --- p.42 / Chapter c. --- Pigmented mycobacteria --- p.43 / Chapter II. --- Vitro Drug Susceptibility Test --- p.43 / Chapter A. --- Mycobacterium tuberculosis --- p.43 / Chapter B. --- Atypical mycobacteria --- p.45 / Chapter 1. --- General characteristics --- p.45 / Chapter 2. --- Sensitivity pattern of different species --- p.46 / Chapter a. --- Mycobacterium kansasii --- p.46 / Chapter b. --- Mycobacterium avium- intracellulare complex --- p.46 / Chapter c. --- Mycobacterium scrofulaceum --- p.47 / Chapter d. --- Mycobacterium terrae complex --- p.47 / Chapter e. --- Mycobacterium fortuitum --- p.47 / Chapter f. --- Mycobacterium chelonae --- p.48 / Chapter III. --- Heavy Metal Tolerance Test --- p.48 / Chapter IV. --- Plasmid in Mycobacteria --- p.48 / Chapter A. --- Mycobacterium tuberculosis --- p.48 / Chapter B. --- Atypical mycobacteria --- p.49 / Chapter 1. --- General characteristics --- p.49 / Chapter 2. --- Correlation between drug resistance and plasmid --- p.50 / Chapter 3. --- Correlation between heavy metal tolerance and plasmid --- p.50 / DISCUSSION --- p.52 / Chapter I. --- Identification of Mycobacteria --- p.52 / Chapter II. --- In Vitro Drug Susceptibility Test --- p.56 / Chapter A. --- Mycobacterium tuberculosis --- p.56 / Chapter B. --- Atypical mycobacteria --- p.60 / Chapter 1. --- Mycobacterium kansasii --- p.61 / Chapter 2. --- Mycobacterium avium- intracellulare complex --- p.61 / Chapter 3. --- Mycobacterium scrofulaceum --- p.62 / Chapter 4. --- Mycobacterium terrae complex --- p.62 / Chapter 5. --- Mycobacterium fortuitum --- p.63 / Chapter 6. --- Mycobacterium chelonae --- p.64 / Chapter III. --- Plasmid Analysis in Mycobacteria --- p.64 / Chapter A. --- Mycobacterium tuberculosis --- p.64 / Chapter B. --- Atypical mycobacteria --- p.66 / SUMMARYS AND CONCLUSIONS --- p.68 / LITERATURE CITED --- p.70 / Chapter APPENDIX - --- Tables --- p.88 / Figures --- p.144

1. The major phospholipids extracted from Bacillus amylolique - faciens were cardiolipin, phosphatidylycerol and phosphatidylethanolamine. 2. The distribution of these phospholipids between the two halves of the cytoplasmic membrane bilayer was studied using phospholipase C ( B. cereus ), phospholipase A2 ( Crotalus ) and the non - penetrating chemical probe trinitrobenzenesulphonic acid ( TNBS ). After treatment of intact protoplasts of B. amylolique - faciens with either phospholipase, approximately 70 % of total membrane phospholipid was hydrolysed ; specifically approximately 90 %, 90 % and 30 % of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold shock treatment were incubated with either of the phospholipases, up to 80 % of cardiolipin was hydrolysed and phosphatidylglycerol and phosphatidylethanolamine were hydrolysed virtually to completion. In intact cells, 92 % of the phosphatidylethanolamine could be labelled with TNBS under conditions in which the reagent did not penetrate the membrane to any significant extent. 3. These results suggest that 70 % of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation or this is required. 4. The fatty acid composition of cells grown at different temperatures was investigated. When cells were grown at 30 ° C, branched - chain saturated fatty acids made up over 80 % of the total fatty acids. Saturated straight - chain fatty acids made up the bulk of the remainder. Less than 1 % of the total fatty acids were unsaturated. Decrease in growth temperature was accompanied by an increase in the ratio of branched to straight - chain fatty acids and a marked increase in the level of unsaturation of branched - chain fatty acids. 5. When cells of this organism, grown at 30 ° C, were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween - 80 to cells caused the critical temperature zone for cold shocking to be significantly lowered. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock. 6. The role of lipids in the process of extracellular enzyme secretion was studied using cerulenin, an antibiotic known to inhibit fatty acid synthesis in microorganisms. Cerulenin inhibited the secretion of alpha - amylase and protease in washed cell suspensions by 80 % and 75 % respectively over 3 hours. The effect was a general one since secretion of all protein species into the medium was drastically reduced by the antibiotic. At the concentration of cerulenin used ( 100 . µ g / ml ), [ 14C ] - acetate incorporation into cellular lipid was inhibited by approximately 50 % but total cellular protein and RNA synthesis were virtually unaffected. The inhibitory effect of cerulenin on alpha - amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens, or various individual pure fatty acids. These results suggest that fatty acid synthesis may be required for protein secretion by this organism. 7. Attempts were made to detect precursors to extracellular enzymes either associated with the cells or in the culture medium, employing immunological techniques. These experiments, however, were not successful. / Thesis (Ph.D.)--Department of Biochemistry, 1979.

The biological world is complex. Communities contain a multitude of interacting species, while populations contain extensive genetic variation. How much complexity must one consider to understand patterns and processes of interest? When are species interactions and community properties shaped by evolution? Conversely, when is evolution altered by community context? I test these questions in a series of experiments with simple microbial communities. The first data chapter investigates the impact of competition on the evolution of phage resistance in bacteria. This work demonstrates that community context can dramatically alter the evolution of resistance to phage. Next I tested the impact of evolution on assembly of a three species community. I demonstrate that evolution can influence the content of a microbial community by altering the process of assembly. Finally, I investigated the evolutionary origin and maintenance of cross-feeding mutualisms. This work suggests that species interactions can enable novel evolutionary pathways, and that evolution can significantly increase the productivity of cross-feeding communities. Jointly these experiments suggest that consideration of the interplay between ecological and evolutionary forces can provide insight into the complexity of the natural world. / text