<div>Host-pathogen interactions are the result of long term evolutionary processes due to the conflicting goals of the host and the infections pathogens in their quest for survival, creating an interplay of co-evolution as various adaptation are acquired by one and then in turned adapted to by the other. Selection of the host’s antimicrobial strategies and the resultant adaptations of infectious microorganisms leads to the development of complex and dynamic relationships ranging from symbiotic to commensal to pathogenic. In an effort to understand the selective process and identify unique mechanisms of antimicrobial defense, sera from 18 species (7 invertebrate, 11 vertebrate) were tested for antimicrobial potential against 20 Gram-negative and 11 Gram-positive bacteria. <i>Alligator mississippiensis</i> sera exhibited the strongest inhibitory potential. A transposon mutagenesis screen performed on the resistant bacterium <i>Serratia marcescens</i> identified several genes, including <i>glnL</i>, as necessary for defense. The <i>glnL</i> gene encodes for the sensory histidine kinase/phosphatase NtrB, controlling the expression of regulatory genes in response to nitrogen limitation. Attenuated growth of the Tn::<i>glnL</i> mutant in the presence of alligator serum and minimal media was rescued with nitrogen supplementation, suggesting the existence of a mechanism for nitrogen limitation as an antimicrobial strategy in alligator sera. Utilization of a <i>Drosophila melanogaster</i> oral model of infection showed that <i>glnL</i> is required for <i>S. marcescens</i> virulence, and nitrogen supplementation rescued the phenotype, as measured by fly mortality and bacterial cfu recovery. S. marcescens, an environmentally ubiquitous Gram-negative bacterium, is an opportunistic pathogen in several species, including alligators and Drosophila. Subsequent <i>in vitro</i> testing of the antimicrobial potential of invertebrate hemolymph utilizing the Tn::<i>glnL</i> mutant showed a nitrogen-dependent growth inhibition of species in the order Dipteria. Combined, these results support a model of evolutionary convergence of nitrogen limitation as an antimicrobial mechanism. This work not only identifies a novel antimicrobial strategy that could be used in the development of therapeutics, and a novel virulence factor in <i>S. marcescens</i>, but has broad mplications for bacterial management and can provide insight into the evolutionary history of host-pathogen interactions.</div>

Most plant pathogens are specialists where only few plant species are susceptible, while all other plants are resistant. Unraveling the mechanisms behind this can thus provide valuable information for breeding or engineering crops with durable disease resistance. A group of Pseudomonas syringae strains with different host ranges while still closely related were thus chosen for comparative study. We confirmed their close phylogenetic relationship. We found evidence supporting that these strains recombined during evolution. The Arabidopsis thaliana and tomato pathogen P. syringae pv. tomato (Pto) DC3000 was found to be an atypical tomato strain, distinct from the typical Pto strains commonly isolated in the field that do not cause disease in A. thaliana, such as Pto T1. Comparing A. thaliana defense responses to DC3000 and T1, we found that T1 is eliciting stronger responses than DC3000. T1 is likely lacking Type III effector genes necessary to suppress plant defense. To test this, we sequenced the genomes of strains that cause and do not cause disease in A. thaliana. Comparative genomics revealed candidate effector genes responsible for this host range difference. Effector genes conserved in strains pathogenic in A. thaliana were expressed in T1 to test whether they would allow T1 to growth better in A. thaliana. Surprisingly, most of them reduced T1 growth. One of the effectors, HopM1, was of particular interest because it is disrupted in typical Pto strains. Although HopM1 has known virulence function in A. thaliana, HopM1 reduced T1 growth in both A. thaliana and tomato. HopM1 also increased the number of bacterial specks but reduced their average size in tomato. Our data suggest that HopM1 can trigger defenses in these plants. Additionally, transgenic detritivore Pseudomonas fluorescens that can secrete HopM1 shows dramatically increased growth in planta. The importance of genetic background of the pathogen for the functions of individual effectors is discussed. T1 cannot be manipulated to become an A. thaliana pathogen by deleting or adding individual genes. We now have a list of genes that can be studied in the future for the molecular basis of host range determination. / Ph. D.

Plants and animals are characterized by intimate, enduring, often indispensable, and always complex associations with microbes. Therefore, it should come as no surprise that when the genome of a eukaryote is sequenced, a medley of bacterial sequences are produced as well. These sequences can be highly informative about the interactions between the eukaryote and its bacterial cohorts; unfortunately, they often comprise a vanishingly small constituent within a heterogeneous mixture of microbial and host sequences. Genomic analyses typically avoid the bacterial sequences in order to obtain a genome sequence for the host. Metagenomic analysis typically avoid the host sequences in order to analyze community composition and functional diversity of the bacterial component. This dissertation describes the development of a novel approach at the intersection of genomics and metagenomics, aimed at the extraction and characterization of bacterial sequences from heterogeneous sequence data using phylogenomic and bioinformatic tools. To achieve this objective, three interoperable workflows were constructed as modular computational pipelines, with built-in checkpoints for periodic interpretation and refinement. The MetaMiner workflow uses 16S small subunit rDNA analysis to enable the systematic discovery and classification of bacteria associated with a host genome sequencing project. Using this information, the ReadMiner workflow comprehensively extracts, assembles, and characterizes sequences that belong to a target microbe. Finally, AssemblySifter examines the genes and scaffolds of the eukaryotic genome for sequences associated with the target microbe. The combined information from these three workflows is used to systemically characterize a bacterial target of interest, including robust estimation of its phylogeny, assessment of its signature profile, and determination of its relationship to the associated eukaryote. This dissertation presents the development of the described methodology and its application to three eukaryotic genome projects. In the first study, the genomic sequences of a single, known endosymbiont was extracted from the genome sequencing data of its host. In the second study, a highly divergent endosymbiont was characterized from the assembled genome of its host. In the third study, genome sequences from a novel bacterium were extracted from both the raw sequencing data and assembled genome of a eukaryote that contained significant amounts of sequence from multiple competing bacteria. Taken together, these results demonstrate the usefulness of the described approach in singularly disparate situations, and strongly argue for a sophisticated, multifaceted, supervised approach to the characterization of host-associated microbes and their interactions. / Ph. D.

As of January 2017, the U.S. poultry industry banned the use of antibiotics and now relies on alternatives such as probiotics to help protect animal health. Although probiotic use is not a new concept in the poultry industry, identifying the best combination of bacterial strains to generate an effective probiotic formula requires further investigation. This study aimed to detect a probiotic product of four bacterial strains (Pedioccoccus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bacillus subtilis) in a feeding trial with broiler chickens. Birds given the probiotic were predicted to show an improved growth performance with the probiotics colonizing the gut. Ninety-six broiler chickens were equally divided into 3 treatment and 3 control pens. During the 25-day experiment, birds were fed a starter diet (days 0-11) and a grower diet (days 12-25). Experimental birds were administered the probiotic product via the drinking water at a concentration of 3.1×104 CFU/ml. Control birds had an equivalent amount of dextrose filler added to their water supply. Feces were collected hourly on day one and daily thereafter. On days 1, 22, and 25 of the experiment, 2 birds from each pen were euthanized for gut sampling. Lumen and mucosa samples were collected from the duodenum, jejunum, ileum, and ceca. Species-specific and strain specific PCR primers were employed for probiotic detection. Wild strains of P. acidilactici, P. pentosaceus, and L. plantarum were detected in the feeds, inhibiting detection of the probiotic strains when using species-specific PCR primers. Strain-specific primers were used to detect the probiotic Pedioccoccus acidilactici and Lactobacillus plantarum strains. B. subtilis was detected in feces within one hour of probiotic administration and was predominantly detected in experimental birds only. Both P. acidilactici and L. plantarum probiotic strains were initially detected in the feces of treated birds within two hours of probiotic administration and again ten days later. Both L. plantarum and B. subtilis were seen only in treated bird gut samples. L. plantarum was predominantly detected in the ceca near the end of the small intestine. P. pentosaceus was observed more often in treated gut samples and P. acidilactici was the least commonly detected probiotic strain. All administered bacteria were rarely seen in mucosa samples. Feed-endogenous P. acidilactici and L. plantarum strains became progressively more detectable in the mucosa along the gastrointestinal tract suggesting gut colonization, however, probiotic strains did not appear to colonize the mucosa of treated birds. Although probiotic strains were no longer detected after product removal, all probiotic strains were detected in feces and gut samples during probiotic administration, suggesting the bacteria can colonize the gut. Probiotic supplementation did not result in significant differences in body weight gain, feed intake, or feed conversion ratio. However, birds growing in a more stressful environment than the carefully controlled experimental set up used here may show probiotic-related effects. This study identified that the probiotic bacteria appeared to survive the gastrointestinal tract, exhibited a transit time of 1-2 hours, could possibly colonize chickens, and localized near the end of the chicken gut.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012