No description available.

Plants rely on innate immunity to perceive and respond to pathogenic microbes. Pathogenic microbes suppress and evade plant immune responses to obtain nutrients and multiply resulting in plant diseases and death. One battleground for the arms race between plants and microbial invaders is located in the leaf intercellular space, specifically between Pseudomonas bacteria and Arabidopsis. This thesis seeks to understand the virulence mechanisms that allow Pseudomonas bacteria to grow within the leaves of Arabidopsis and how the plant immune response reduces pathogen growth and reproduction. Some plant pathogens produce specific extracellular polysaccharides to potentially enhance pathogenicity during infection of plants. The objective of this thesis is to understand the importance of biofilms for Pseudomonas success and determine if Arabidopsis suppresses biofilm formation as part of the plant immune response. It was hypothesized that biofilm formation contributes to Pseudomonas success in planta and Arabidopsis suppresses biofilm formation during PAMP-Triggered Immunity (PTI) to reduce bacterial growth. Wild-type plants and defense mutants were infiltrated with flg22 or mock (water) treatments to induce or mock-induce PTI in plants, followed by observing GFP-expressing Pseudomonas via florescence microscopy to determine if biofilm-like aggregate formation was occurring. In vivo studies in this thesis indicate that biofilm-like aggregate formation contributes to bacterial success during Arabidopsis infection. Additionally, the phytohormone, salicylic acid (SA), accumulates in leaf intercellular spaces of resistant plants during PTI that suppresses biofilm formation, suggesting that SA acts as an anti-microbial and anti-biofilm agent that contributes to the suppression of pathogen growth during plant defense. / Thesis / Master of Science (MSc)

Biofertilizer (or microbial soil inoculants) may be used to reduce current fertilizer inputs (organic or conventional methods), while maintaining or improving crop plant growth and yield. Pseudomonas putida is a plant-growth promoting rhizobacterium (PGPR) that solubilizes inorganic phosphorous or mineralizes organic phosphates, produces siderophores (enhancing Fe availability for plants, reducing Fe access to pathogenic fungi), and is known to improve plant growth by assisting with nutrient availability, synthesis of plant hormones (indole acetic acid regulation production and/or degradation, or ethylene regulation through aminocyclopropane carboxylate deaminase activity), and acts as a biological control of several plant pathogens and pests (Rhizoctonia solani, Bemisia tabaci). Recommendations for inoculum population density, application timing, and species of PGPR, vary mainly based on geographic and weather conditions, and their relationship to each horticultural crop, which needs to be better understood. Two studies were conducted in 2014 and 2015 at the Southern Illinois Horticulture Research Center to determine the optimum application timing and dosage of Pseudomonas putida strains 17-29 and G11-32 to improve plant growth and yield on two important horticultural crops: field grown ‘Revolution’ bell peppers and greenhouse grown ‘Rocky Top’ determinate tomatoes. Field pepper results indicated that the inoculum population density increased early-season vigor, plant height and stem caliper (P < 0.05), as well as late-season plant vigor, height (cm), leaf chlorophyll index (SPAD), and stem caliper (mm) (P < 0.05) for inoculum population density. Although early harvests (first two harvests) fruit yield increased with rhizobacteria inoculum population density (P < 0.05) for total fruit weight (2014, 2015) and number (2014), late-season fruit yields (last three harvests) were not affected. Combined fruit yield total weight (all five harvests) also increased (P < 0.05) by inoculum population density in the order: 10^0 < 10^3 < 10^5 < 10^7 < 10^9. It appears that higher early-season and combined harvest yields were higher resulting from increasing inoculum population densities were greater due to increased early season growth from the bacterial treatments during plug growth phase. Tomato results indicated that inoculum population density increased early growth and late growth vigor, height (cm), leaf chlorophyll index (SPAD), leaf number, flower number, fresh leaf weight (g), fresh stem weight, and dry leaf weight (P < 0.05). Tomato growth was effected by inoculum population density treatment however, yields differences were not observed.

No description available.

Life on Earth is incredibly diverse. The process of diversification that gives rise to this diversity is not the same for all lineages. Diversification is often driven by ecological opportunity. Pseudomonas aeruginosa is an opportunistic pathogen present in a variety of environments that causes chronic lung infections in cystic fibrosis (CF) patients. It diversifies rapidly within the CF lung and CF lung-like environments. Here we aim to assess both ecological and genetic factors in diversification of several strains of P. aeruginosa. We evolved 12 replicate populations of 8 different strains of P. aeruginosa in a nutritionally complex (LB) and simple environment (MIN) for 750 generations. We then measured diversity over time by observing the number of colony morphologies in each population every 250 generations. We also measured competitive fitness relative to the ancestor for endpoint populations. To provide a more complete analysis, phylogeny was factored into our statistical models. First, we found no significant differences in diversification between populations evolved in LB versus MIN media. Ancestor population size had no significant effect on diversification. We found that in both selection environments, CF strains diversified less than environmental strains, but this difference was marginally significant and only present when comparing these two niches directly and excluding acute strains. Finally, we found no correlation between gains in fitness and endpoint diversity. Our results suggest that diversification is limited by niche specialization (domestication) of P. aeruginosa to the CF lung.

Aspartate transcarbamoylase (ATCase) is encoded by the pyrBI genes in E. coli. Expression of these genes is reduced four-fold by attenuation when grown on uracil. Using plasmid, pRO1727. the pyrB and the pyrBI genes from E. coli were cloned into a P. putida pyrB auxotroph. A recombinant pyrB gene was recovered that encoded a functional hybrid ATCase with a molecular weight of 470 kDa.

Even though it has been decades since antibiotics were put into widespread use, bacterial infections are a worsening source of morbidity and mortality worldwide. This is partially due to the formation of biofilms. Biofilms are populations of microbial cells embedded in self-produced matrices and their formation can enhance survival of the pathogen in the host. Pseudomonas aeruginosa is a major cause of acute and chronic infections and an excellent model for the study of opportunistic, biofilm-based infections. It produces a plethora of virulence factors and we do not fully understand how it harms the host. This thesis investigates the synthesis and characteristics of the Refractile-body (R-body), a newly identified P. aeruginosa virulence factor and potential roles of this virulence factor during host colonization. R-bodies are large proteinaceous polymers that are produced as a coiled ribbon but can extend to form a spear-like structure that is longer than a bacterial cell. Further, the R-body is produced stochastically and the producing minority is thought to contribute to success of the population through altruistic suicide. The purpose of this thesis is to characterize yet another virulence factor in the arsenal of the notorious pathogen P. aeruginosa. Further, the capacity for R-body production is present in diverse bacteria, and characterization of its function could be pertinent for our understanding of other bacteria with roles in medicine, agriculture, and industry. In Chapter 1, I introduce concepts from the fields of bacterial infectious disease, population biology and gene expression to provide context for my research findings on the R-body. In Chapter 2, I describe the discovery of R-body polymers in the P. aeruginosa PA14 biofilm. Using mass spectrometry analysis, I identified a novel P. aeruginosa R-body protein absent in the Caedibacter taeniospiralis and Azorhizobium caulinodans genomes, two bacteria for which R-body production had previously been described. Further, results in the chapter elucidate the role of R-bodies in P. aeruginosa PA14 colonization in the plant and virulence in the nematode hosts. The work described in Chapter 3 focuses on the transcription factor RcgA, which is required for R-body production. The gene encoding RcgA lies in a cluster and is co-expressed with R-body structural genes. Using established genetic tools, I asked the question, “what signal does RcgA sense?” I found that RcgA binding to a cyclic nucleotide is necessary for its function in turning on R-body genes. I present data in Chapter 3 and 4 that sheds light on the regulatory logic of R-body production in P. aeruginosa. Specifically, using single-cell resolution methods, I have been able to characterize the impact of various genes on stochasticity of R-body production in the population. Data presented in these chapters are another example of the importance of studying heterogeneity and stochasticity of virulence factor expression in the population. Taken together, the work in this thesis provides an expanded and multifaceted understanding of a fascinating virulence factor found across bacterial phylogeny. The R-body produced by P. aeruginosa, a notorious human pathogen, is unique in its makeup and should be further characterized. This work also underscores the necessity of studying bacterial pathogenicity in the context of the biofilm lifestyle.

Type IV pili (T4P) are long, thin, flexible surface appendages used by various bacteria for surface adhesion, cell-cell aggregation, DNA uptake, biofilm formation and motility. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, and uses T4P as a key virulence factor to infect immunocompromised individuals. Four subcomplexes make up a functional T4P system in P. aeruginosa and the role of the alignment subcomplex is to physically connect the outer membrane pore with the inner membrane motor, allowing for efficient extrusion of the pilus fibre from the cell. Two alignment subcomplex proteins, PilN and PilO, form heterodimers and are required for proper function of the system. These proteins may be able to transduce signals between various T4P components to indicate extension and/or retraction of the pilus fibre. This thesis focused on characterization of the interaction interfaces between PilN and PilO, and on understanding the dynamics required for proper function of the system. We show that although PilN and PilO make extensive interaction contacts throughout their lengths, single point substitutions at key residues can successfully disrupt the function of the T4P system. Crosslinking PilN and PilO as homo- or heterodimers can disrupt motility and surface piliation, indicating that interfaces between these proteins must be dynamic to allow proper T4P function. A high resolution X-ray crystal structure of PilO was solved and exhibits new structural features previously unidentified. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, as well as defining a role for these proteins in extension and retraction. / Dissertation / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is an opportunistic bacterium, able to infect individuals with weakened immune systems. It attaches to and moves along surfaces using long, thin, sticky, retractable fibres known as type IV pili. Similar to a grappling gun, a functional type IV pilus system requires four subcomplexes working in unison to allow for the extension, adherence, and retraction of pilus fibres, which pulls the cell forward towards the point of attachment. Two key proteins, PilN and PilO, are bound to each other and allow for efficient extension and retraction of the pilus fibre. This study focused on characterization of the interactions of PilN and PilO, and on understanding whether dynamic rearrangements of the interfaces between these proteins is required for proper function of the system. We show that although these proteins have extensive interaction interfaces, single residue substitutions in either of them can disrupt the ability of the bacteria to properly extend and/or retract their pili. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, providing information that might allow disruption of these interfaces to block bacterial attachment or motility, both of which are important for infection.

Type IVa pili (T4aP) are long, retractile, filamentous, surface appendages involved in cellular surface adhesion, biofilm formation, DNA uptake, and a unique form of motility called ‘twitching’. They are a critical virulence factor in a number of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, a major cause of hospital-acquired infections. T4aP function is controlled by a number of different regulatory proteins and systems. A putative chemosensory system termed ‘Chp’, controls levels of the second messenger molecule cyclic adenosine monophosphate (cAMP). cAMP works with a cAMP receptor protein called Vfr to control expression of ~200 virulence genes, including those that are required to make T4aP. cAMP levels are regulated by proteins outside the Chp system, including the bitopic inner membrane protein, FimV. This study examines the role of the Chp system and FimV in T4aP regulation. Both proteins are required for regulation of cAMP levels, while the Chp system also has a cAMP-independent role in regulating twitching. FimV has been shown to regulate cAMP levels, possibly connecting to the Chp system through a scaffold protein, FimL. We present the structure of a conserved cytoplasmic region of FimV, and show that this region is required for connecting FimV to the Chp system. We also characterize the cAMP-independent role of FimV, confirming that it is distinct from that of the Chp system, and is involved in localizing T4P regulatory proteins. We also provide evidence that the cAMP-independent role of the Chp system is to mediate the balance between T4P extension and retraction, possibly through denoting the ‘front’ of a motile cell. Together, these data help to resolve the cAMP-independent and –dependent pathways controlling twitching motility. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a bacterium that causes infection in people with weakened immune systems. One key factor it uses to cause infection is the type IVa pilus (T4aP), a filamentous appendage displayed on the cell surface. T4aP can repeatedly extend and retract, and are involved in attachment to host cells, and movement along surfaces. When T4aP cannot extend or retract, the bacteria cannot cause infection. Many proteins work together to control T4aP function – this study focuses on two of them. They have one overlapping function, controlling levels of a signalling molecule needed to make T4aP. We also show that they have a second, non-overlapping function. One is involved in controlling the extension/retraction balance, possibly by marking the front of a cell, while the other may localize pilus-related proteins within a cell. This work helps us understand how P. aeruginosa makes T4aP, and provides information helpful to understanding control of virulence.

The transition from unicellular to multicellular life is captivating because free-living individuals become complex, coordinated assemblages that display unique properties and behaviors. It is a transformative step in biology that optimizes survival and resource utilization, especially in fluctuating environments. In microbiology, this multicellular organization assumes an intriguing form known as biofilms. Bacterial biofilms, assemblages of cells encased in a self-produced matrix, are sophisticated structures that provide protection from environmental challenges. The emerging understanding of biofilms reveals that bacteria within them do not exist as passive, isolated entities. Instead, they display spatial organization, physiological differentiation, and even metabolic interactions such as cross-feeding. The pathogenic bacterium Pseudomonas aeruginosa, which is a common cause of biofilm-based infections and a popular model organism, has been shown to form metabolic subpopulations and differentially regulate gene expression across depth in biofilms. However, one open question is the nature of this cellular arrangement in P. aeruginosa biofilms, the mechanisms governing it, and its physiological ramifications. My thesis addresses the overarching question: Does cellular arrangement in P. aeruginosa biofilms influence nutrient distribution, metabolic activity, antibiotic tolerance, and metabolic cross feeding? Through the use of paraffin embedding, thin-sectioning, and confocal microscopy, I delve deep into the biofilm, particularly in the z-direction, byproducing high-resolution images that provide insights into the three-dimensional structure and dynamics of these bacterial communities. The first chapter, serving as the foundation of this exploration, provides an introduction of the principles of multicellularity. It draws attention to the hallmarks of multicellularity, encompassing metabolic cross-feeding, protective advantages, and labor specialization while also shedding light on its challenges. In the context of multicellularity, biofilms are introduced, emphasizing the formation of bacterial biofilms, their environmental and medical implications, and specifically highlighting the importance of P. aeruginosa biofilms for understanding microanatomy and physiology. Chapter 2 presents the crux of our exploration, underlining how cellular arrangement directly impacts metabolic activity and antibiotic tolerance in P. aeruginosa biofilms. A striking observation was the presence of vertical, clonal striations, suggesting the presence of an organized architecture within mature biofilms. Mutants with disordered cell arrangements, particularly in O-antigen attachment, showed altered patterns of nutrient distribution and metabolic activity in addition to distinct patterns of antibiotic- induced cell death. Such findings build on prior knowledge by illuminating the intricate relationships between biofilm anatomy, metabolic differentiation, and drug tolerance. Chapter 3 introduces the use of light-sheet microscopy for live imaging of pellicle biofilms, which offers a real-time window into biofilm development and cellular dynamics. In Chapter 4, the narrative takes a broader perspective, focusing on the influence of various carbon sources on cellular arrangement. It introduces the presence of metabolic cross-feeding among different biofilm subpopulations and hints at the potential relationship between cell arrangement and heterogeneous metabolic activity patterns. The work in this thesis reveals that the arrangement of cells within P. aeruginosa biofilms determines metabolic outcomes, antibiotic responses, and potential cross- feeding interactions. In a world where biofilm-related infections account for an alarming 80% of persistent bacterial infections, understanding biofilm microanatomy has implications for therapeutic strategies and possibly reshaping our battle against antibiotic tolerance. A more detailed picture of the relationship between cell arrangement, physiological differentiation, and metabolic cooperation within biofilms has the potential to provide inroads toward new approaches to combating these recalcitrant structures.