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In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment. A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta. Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens.

E. coli makes use of global transcriptional regulators to survive ever-changing environmental conditions. The leucine-responsive regulatory protein (Lrp) is one such global regulator within E. coli . With its co-regulator leucine, Lrp can activate or repress target genes. Many of these regulatory interactions are direct, with Lrp binding to its cognate DNA to perform its regulation. Lrp can also act indirectly by regulating a gene that affects the expression of other genes or proteins. The role of Lrp in E. coli defies simple classification, but Lrp is thought to mediate transitions between rich and poor growth media by controlling expression of amino acid biosynthetic, catabolic and transport genes. Two-dimensional electrophoresis and microarrays were employed to better understand the physiological role of Lrp. New members of the Lrp regulon were identified on 2D-gels by noting proteins spots whose expression was altered when comparing wildtype (W3110) and isogenic lrp::Tn 10 strains of E coli. These protein spots were unambiguously identified by mass spectrometry. The newly identified members of the regulon identified by 2D-gels electrophoresis range in function from amino acid biosynthesis to quorum sensing and include argF, dapB, deoD, fbaB, guaB, fur, and luxS. Regulation of these genes by Lrp was confirmed using translational fusions. Furthermore, interaction of Lrp with the promoter regions of the corresponding proteins was tested using gel mobility shift assays. Lrp has been found to interact directly with the promoter regions of four (argF, dapB, fbaB and luxS) out of the seven new members. DNA microarrays were used to carry out a systematic study of wt and lrp:;Tn10 strains at the mRNA level. The arrays identified 427 Lrp-responsive genes, of which over 80% (340) were not previously recognized as members of the Lrp regulon. Surprisingly, a large number of these genes were Lrp-responsive whether or not exogenous leucine was present. These Lrp-responsive genes represent 10% of the E. coli genome and cluster within distinct functional classifications. Lrp is postulated to be involved in new physiological roles including iron uptake, nucleoside catabolism, acid shock, and entrance into stationary phase. Additional Lrp-responsive genes have been identified in amino acid transport, anabolism and catabolism, and include genes involved in the synthesis of fimbriae and flagella.