You are here
Search results
(1 - 9 of 9)
- Title
- Cloning of the polysaccharide depolymerase gene of bacteriophage pea1 (h) and its expression in Erwinia amylovora
- Creator
- Hartung, John Stephen
- Date
- 1985
- Collection
- Electronic Theses & Dissertations
- Title
- Cyclic di-GMP dependent regulation in the plant pathogenic bacterium Erwinia amylovora
- Creator
- Castiblanco Mosos, Luisa F.
- Date
- 2016
- Collection
- Electronic Theses & Dissertations
- Description
-
Bacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized...
Show moreBacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized intracellularly and degraded by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. This nucleotide signaling molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures, and cell differentiation. Signal transduction and phenotypic modulation is determined by binding of c-di-GMP to specific downstream receptor molecules.Erwinia amylovora uses motility and the hypertensive response and pathogenicity- (Hrp-) type III secretion system (T3SS) during initial phases of plant infection. Once inside the xylem, this pathogen aggregates due to the production of exopolysaccharides (EPS) and attachment structures forming a biofilm that ultimately plugs the xylem vessels. Although c-di-GMP has been shown to be an important intracellular signal in several plant pathogenic bacteria, the importance of this molecule in Erwinia amylovora has not been previously investigated. This doctoral research explores the role of c-di-GMP in the genetic regulation of key cellular processes associated with pathogenesis in E. amylovora. Five active DGC enzymes (EdcA, EdcB, EdcC, EdcD and EdcE) were identified in this bacterial pathogen. Phenotypic analyses demonstrated that c-di-GMP positively regulates the biosynthesis of both cellulose and amylovoran, positively regulates biofilm formation, and represses motility. Disease assays demonstrated that c-di-GMP negatively regulates virulence in these infection models.The exopolysaccharides amylovoran and levan and attachment structures such as fimbriae, type IV pili and curli, have been demonstrated to be critical for the formation of a mature biofilm in E. amylovora. The results presented in this work demonstrated that c-di-GMP binds to the receptor protein BcsA, the cellulose synthase subunit, and activates the biosynthesis of cellulose in E. amylovora at a post-translational level. In addition, using SEM and confocal microscopy, it was demonstrated that cellulose is a main component of the biofilms formed by E. amylovora in vitro and in the host. Gene overexpression and site-directed mutagenesis analyses, suggest that BcsZ, an endoglucanase, is also required for cyclic di-GMP activation of cellulose biosynthesis and biofilm formation.Further investigation into the mechanisms of c-di-GMP dependent regulation of virulence, with a special emphasis on the regulation of hrp-T3SS gene expression, revealed that high intracellular levels of c-di-GMP via DGC overexpression lead to a significant reduction in gene expression for hrpL, the hrp alternative sigma factor, and dspE. C-di-GMP binding assays suggest that HrpS, the σ54 dependent transcriptional regulator of hrpL, is a candidate receptor of c-di-GMP. Moreover, the results presented in this work indicate that HrpS can bind to other guanosine-containing signaling molecules including the c-di-GMP degradation product pGpG. This work provides an overview of some of the molecular mechanisms for c-di-GMP-dependent regulation on the main pathogenicity determinants in E. amylovora, in order to orchestrate pathogenesis and successfully cause disease.
Show less
- Title
- Fungicide resistance analysis of major tree fruit pathogens in Michigan
- Creator
- Lesniak, Kimberley E.
- Date
- 2016
- Collection
- Electronic Theses & Dissertations
- Description
-
Chemical and/or antibiotic resistance management is critical in order to reduce the progression or complete loss of a product while effectively controlling plant pathogen populations. This dissertation focuses on the observation for control and resistance management of four major worldwide fruit tree pathogens: Venturia inaequalis (apple scab), Monilinia fructicola (American brown rot), Erwinia amylovora (Ea; fire blight) and Pseudomonas syringae pv. syringae (Pss; bacterial canker). Chemical...
Show moreChemical and/or antibiotic resistance management is critical in order to reduce the progression or complete loss of a product while effectively controlling plant pathogen populations. This dissertation focuses on the observation for control and resistance management of four major worldwide fruit tree pathogens: Venturia inaequalis (apple scab), Monilinia fructicola (American brown rot), Erwinia amylovora (Ea; fire blight) and Pseudomonas syringae pv. syringae (Pss; bacterial canker). Chemical fungicides have been and are still heavily relied on for management of the fungal plant pathogens V. inaequalis and M. fructicola. Single-site fungicides such as the quinone-outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) have shown to provide excellent control but can also select for resistant pathogen populations in a relatively short time period, as in the case for both V. inaequalis and M. fructicola. Adequate control of bacterial pathogens, such as Ea and Pss has been a challenge for pome and stone fruit growers for decades. Antibiotics have been intensively used for the management of Ea while only copper formulations are registered for Pss. The current research implicates that orchards in Michigan and in other states cannot further rely on QoI fungicides for apple scab control. Further investigation of peach and cherry orchards indicates that SDHI applications have shifted Michigan populations toward resistance. Results from studies performed with Ea and Pss indicate that non-chemical applications such as acibenzolar-S-methyl (ASM) and phosphate salts have a potential to reduce the use of antibiotics and copper for control in seasons when disease pressure is low to moderate.
Show less
- Title
- Resistance against Erwinia amylovora induced in apple trees by acibenzolar-s-methyl
- Creator
- Maxson, Kimberly Lynn
- Date
- 2000
- Collection
- Electronic Theses & Dissertations
- Title
- Role of waaL and umuDC in Erwinia amylovora EA1189 in oxidative stress and ultraviolet radiation survival
- Creator
- Berry, Matthew
- Date
- 2009
- Collection
- Electronic Theses & Dissertations
- Title
- Roles of hfq-dependent srnas in e. amylovora regulation of virulence
- Creator
- Schachterle, Jeffrey Kent
- Date
- 2019
- Collection
- Electronic Theses & Dissertations
- Description
-
Erwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III...
Show moreErwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. Complex regulatory mechanisms have evolved in E. amylovora that occur at the transcriptional, post-transcriptional, and post-translational levels to control these virulence factors. In my work, I analyze the role of small RNAs (sRNAs) as post-transcriptional regulators of virulence-associated traits in E. amylovora.The Hfq chaperone protein stabilizes sRNAs in the cell, allowing them to interact with and regulate mRNA targets. An hfq mutant differs from wild-type cells in several virulence-associated phenotypes including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. E. amylovora encodes at least 40 Hfq-dependent sRNAs; in my work, I have systematically made deletion mutants of each sRNA singly, as well as constructed inducible expression vectors for each sRNA. Screening of this sRNA library has shown that several sRNAs contribute to regulation of each virulence phenotype, indicating complex regulation of the traits assessed. Of particular interest, the ArcZ sRNA regulates several of the virulence-associated traits we have assessed, and an arcZ deletion mutant loses virulence in both immature pear and apple shoot infection models.Flagellar motility, which enables E. amylovora cells to swim through flower nectar to invade natural openings in host flowers, is regulated by ArcZ. We have shown that ArcZ regulates motility by regulating the flagellar transcription factor FlhD at both the transcriptional and post-transcriptional levels. Because the ArcZ regulation of FlhD at the transcriptional and post-transcriptional levels has a contradiction in sign, we searched for additional layers of regulation between ArcZ and FlhD. We did so by conducting a transposon screen in the arcZ mutant background for suppressor mutants that restored flagellar motility. This screen yielded as the most common suppressor mutation the leucine responsive regulator protein (Lrp), a global transcription factor known for regulation of amino acid metabolism. We have found that Lrp not only acts as a regulator of flagellar motility between ArcZ and FlhD, but that it also reverses the regulatory effects of arcZ deletion on amylovoran and levan production, as well as biofilm formation. Our work shows that Lrp is a novel virulence regulator that plays an important role in regulating several virulence-associated traits in conjunction with the sRNA ArcZ.Transcriptomic comparison between the arcZ mutant and wild-type cells confirmed that ArcZ regulates several genes known to also be regulated by Lrp, and also indicated that ArcZ regulates several genes involved in mitigating the threat of reactive oxygen species, including genes encoding a catalase, a thiol-peroxidase, and a peroxiredoxin. We found that catalase makes the greatest contribution to diminishing the threat of exogenous hydrogen peroxide. Additional analysis suggests that ArcZ participates in regulation with an oxidative sensing transcription factor network that includes the transcription factors ArcA, Fnr, and Fur.This work shows that several sRNAs make small contributions to virulence trait regulation, and that a few sRNAs, like ArcZ, make major contributions to E. amylovora virulence. ArcZ regulates several virulence-associated traits through the global transcription factor Lrp, which we have found to be a novel virulence regulator. ArcZ also regulates genes involved in mitigating the threat of reactive oxygen species, which can protect E. amylovora cells from host defenses during infection. Thus, ArcZ plays an integral role in modulating phenotypic expression during fire blight disease progression that enables E. amylovora to successfully colonize and infect host plants. Mechanistic understanding of E. amylovora gene regulation moves us closer to understanding weaknesses that can be exploited for development of novel disease control strategies.
Show less
- Title
- Secretion signals and chaperone function in Erwinia amylovora
- Creator
- Triplett, LIndsay R.
- Date
- 2009
- Collection
- Electronic Theses & Dissertations
- Title
- Studies on the nutrition of Bartlett pear trees (Pyrus dommunis L) and fire blight (Erwinia amylovora, Burr.)
- Creator
- Lewis, Lowell Nelson
- Date
- 1960
- Collection
- Electronic Theses & Dissertations
- Title
- The mechanisms of streptomycin resistance in Erwinia amylovora
- Creator
- Chiou, Chien-Shun
- Date
- 1994
- Collection
- Electronic Theses & Dissertations