Thesis (Ph. D.)--Ohio State University, 2004. / Document formatted into pages; contains xxxiii, 421 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Dec. 2.

Center rot of onion, caused by Pantoea ananatis, was first described in the USA, in 1997. P. ananatis is seed-borne in onions and it was suggested that it was introduced into the USA on infected seed lots from South Africa. Center rot has not been observed in South Africa and it was essential to determine if P. ananatis is present in local onion seed. Colonies resembling those of P. ananatis were isolated from four South African seed lots on PA 20, a new semi-selective medium. Pathogenicity tests demonstrated that the South African and America strains induced the same symptoms on onion. Phenotypic and genotypic analyses identified the strains from seed as P. ananatis. In 2004/2005, an unreported disease of maize, brown stalk rot, was observed on commercial fields in South Africa. The representative strains induced disease symptoms similar to those observed in the field. The phenotypic and genotyping tests showed that the strains belonged to the genus Pantoea and separated them into two groups. The first group was identified as P. ananatis. The F-AFLP genomic fingerprints generated by the second group of strains, were distinctly different from those generated by known Pantoea species. To resolve the taxonomic position of Pantoea isolated from onion and maize, sixty-seven strains were subjected to a polyphasic study. The methods used included phenotypic characterisation, genomic fingerprinting, 16S rRNA gene sequence analysis and DNA-DNA hybridisation. The results revealed that the strains belong to three different species within the genus Pantoea: P. ananatis, P. vagens and a novel species, Pantoea allii sp. nov. / Thesis (PhD (Microbiology))--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Yes / Pantoea stewartii is known to be the causative agent of Stewart's wilt, which usually affects sweet corn (Zea mays) with the corn flea beetle as the transmission vector. In this work, we present the whole-genome sequence of Pantoea stewartii strain M009, isolated from a Malaysian tropical rainforest waterfall. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01 no. A-000001- 50001 and UM C/625/1/HIR/MOHE/CHAN/14/1 no. H-50001-A000027)

Doctor of Philosophy / Department of Entomology / Marcelo Ramalho-Ortigao / Kun Yan Zhu / Microbial communities residing within the midgut of insect vectors play a critical role in the response to various zoonotic and human pathogens, and can directly alter the development and survival of the insects. Sand flies are the primary vector of Leishmania, the causative pathogen of leishmaniasis, a neglected tropical disease. Sand flies acquire many microbes from the soil where immature stages develop until emergence as adults. Gram-negative Pantoea agglomerans and gram-positive Bacillus subtilis are two bacteria commonly associated with sand fly populations. Here, I demonstrated that an EGFP- and a GFP-expressing version of these two bacteria localize to different compartments of the midgut; a phenomenon that is achieved, in part, to pH differences found across the length of the gut. Additionally, P. agglomerans is able to selectively induce midgut epithelial apoptosis while B. subtilis does not. This is accompanied by differential immune and homeostasis responses to both bacteria highlighted by immune pathway suppression via the Poor Immune Response upon Knock-in (Pirk) gene. These effects may actually be representative of a broader type of response to bacterial infection that might be present across several insect species. Finally, I demonstrated that during metamorphosis the sand fly relies, at least in part, upon the activation of multiple genes from the autophagy pathway to aid in generating adult tissues. More specifically, I demonstrate, using microscopy, the presence of ATG6 in the cytoplasm of developing midgut epithelial cells of the sand fly pupae.

Next generation of sequencing (NGS) technologies have taken life science research into a new era. With the rapid advances in these technologies and the associated reduction in overall costs, the sequencing and assembly of genomes have come within reach of most laboratories. Studies related to the evolution, ecology and biology of an organism now rely heavily on genomic data and obtaining a genome sequence has become an essential resource for the rapid progress and success of these studies. Pantoea ananatis is recognised as an emerging but rather unconventional pathogen capable of infecting a wide range of different hosts. Numerous plants of agricultural and economic importance including maize, rice, onion, pineapple, melon, sudan grass and Eucalyptus trees have been affected. With the outbreak of P. ananatis in a South African Eucalyptus nursery in 1998, it was realised that very little is known about this pathogen. A better understanding of the pathogenicity, metabolism and ecology of the bacterium is required to develop strategies for the control of the disease. During this study, the genome sequence of P. ananatis strain LMG 20103 was obtained using the Roche 454 technology. To aid in the assembly of this Eucalyptus pathogen’s genome sequence, the type strain of P. ananatis LMG 2665 was also sequenced using Illinima’s Genome Analyzer (GA). A draft assembly of P. ananatis LMG 20103, consisting of 117 contigs, was generated after optimization of the Newbler assembly parameters and comparison with other genome assemblies and genomes. This study demonstrated that the assembly could be completed using both in-vitro, and in-silico approaches such as contig scaffolding, gap closure with conventional PCR reactions and sequencing, manual curation and automated genome annotation. The final complete genome consisted of a 4 386 227 bp chromosome and a 317 146 bp mega-plasmid. With the complete genome sequence available, the reconstruction of metabolic network of P. ananatis LMG 20103 was attempted using two pathways reconstruction pipelines namely, Pathway Tools and Model SEED. It was found that missing metabolic reactions and incomplete pathways in the draft metabolic networks were mainly caused by incorrect gene annotations or bioinformatic errors during the automated network reconstruction. These two pipelines differed substantially in the way network reconstruction is undertaken. Performing a comparison between the two proposed networks, annotation errors could be detected and corrected. Although some improvement could be made to the predicted network further experimental data is still required to improve the accuracy of the draft metabolic network. Despite the amount of effort and cost, it is believed that the complete genome and a draft metabolic network of P. ananatis LMG 20103 will be a valuable resource for many subsequent studies to investigate the evolution and biology of this emerging plant pathogen. This information will be essential for the development of strategies to predict and control future disease outbreaks associated with this pathogen. / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Introduction: Pantoea agglomerans is a Gram negative ubiquitous bacteria commonly isolated from plant surfaces, seeds, fruits and animal/human feces usually introduced to human by ingestion of infected fruits/vegetables, thorn pricks and gastrointestinal translocation in lack of stomach acidity. However, the pathogen can also cause opportunistic human infection especially when the immune system is impaired. The aim of this case report is to investigate clinical features in a patient with P. agglomerans bacteremia and bring attention the opportunistic infection by this rare bacteria. Case presentation: We present a case of 57 year old caucasian lady with past medical history of Chronic Obstructive Pulmonary Disease, Atrial fibrillation, Immunoglobulin (IgG) deficiency, recurrent pneumonia, urine infection, oral/vaginal candidiasis, Gastro-esophageal reflux disease who presents with one week history of increased shortness of breath, chest tightness and productive cough without fever/chills. She also had high INR of 4.7 (target 2-3) despite taking normal dose of warfarin. She denies plant exposure. Her vitals were stable, saturation maintained with oxygen supplementation. Chest exam revealed very poor air entry bilaterally suggesting exacerbation of COPD. Oral thrush was present. Recent IgG level within last 6 months was low. Blood culture grew Pantoea agglomerans, pan-sensitive to most of the antibiotics. Chest X ray, CT scan abdomen and urine studies could not localize the source of infection. She was treated with Ceftriaxone, INR normalized to therapeutic range and she improved to baseline after 10 days of treatment. Discussion and conclusion: P. agglomerans is a rare cause of bacteremia which usually presents as fever, chills and general toxicity, however could also present as a cause of exacerbation of chronic diseases. Spontaneous infection can occur in a immunocompromised host, however the pathogen is of low virulence. The link between upper GI symptoms along with antacid receipt and spontaneous P. agglomerans infection could be possible, however needs further study. Hence, P. agglomerans should be considered one of the possible cause of spontaneous bacteremia in a immunocompromised host.

No description available.

Pantoea stewartii subsp. stewarti is the causative agent of Stewart's wilt disease in maize. Disease symptoms develop after the bacteria grow to high cell densities in the plant xylem and secrete an abundance of exopolysaccharide (EPS). EPS production is regulated by quorum sensing. Two regulatory proteins are key to the process of quorum sensing, the LuxI and LuxR homologues EsaI and EsaR. Most LuxR homologues function as activators of transcription in the presence of their cognate acylated homoserine lactone signal (AHL). EsaR utilizes an AHL-response opposite of the majority of the LuxR homologues. EsaR represses EPS production at low cell densities. However, at high cell densities when high concentrations of AHL are present, EsaR is inactivated and derepression of EPS production occurs. The mechanism that enables EsaR to respond to AHL in a manner opposite to that of most LuxR homologues remains elusive. A comparative study of EsaR and the well characterized quorum-sensing regulators LuxR from Vibrio fischeri and TraR from Agrobacterium tumefaciens was initiated. Previous studies demonstrated that in the absence of AHL, EsaR retains the ability to function as a weak activator of the lux operon in recombinant Escherichia coli. This thesis research further characterized the role of EsaR as an activator. Variant forms of EsaR with deletions or single residue substitutions were generated and their ability to regulate transcription was examined in vivo. Furthermore, a native EsaR-activated promoter has been identified, which controls expression of a putative regulatory sRNA in P. stewartii. It is apparent that EsaR functions as a transcription factor at low concentrations of AHL as demonstrated by its ability to inhibit EPS production. At high concentrations, the AHL appears to bind and cause a conformational shift in the protein leading to its inactivation. The second goal of this study was to further elucidate the mechanism by which AHL regulates EsaR. Pulse-chase experiments demonstrated that EsaR is resistant to proteases with or without AHL in vivo. Limited proteolytic digestions in vitro suggest that the protein does undergo conformational changes in response to AHL. Gel filtration chromatography, sucrose gradient ultracentrifugation, and cross-linking experiments proved that this conformational change does not impact the multimeric state of EsaR. To better understand the mechanism of regulation by AHL, the final goal of this project was to examine the interactions which result in EsaR-responsiveness to AHL. Several individual amino acid substitutions were identified that cause EsaR to function in an AHL-independent manner, by which variants retain the ability to bind and block gene expression in the presence of AHL. These residues have been mapped onto a homology model of EsaR and their role has been examined in vitro. The ability of these EsaR* variants to bind AHL and an analysis of the effects individual mutations have on the overall conformation of the protein was performed. Overall this study has revealed several unique aspects of the quorum-sensing system in P. stewartii whereby gene expression is regulated at both low and high cell density. Studies were also initiated to examine the mechanism of AHL-responsiveness of EsaR. The mechanism by which AHL modulates most LuxR homologues remains elusive. The ability to purify EsaR +/- its cognate AHL may prove critical in elucidating this mechanism. / Ph. D.

The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR. / Master of Science

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science