scholarly journals Mutation of theErwinia amylovora argDGene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

2014 ◽  
Vol 80 (21) ◽  
pp. 6739-6749 ◽  
Author(s):  
Laura S. Ramos ◽  
Brian L. Lehman ◽  
Kari A. Peter ◽  
Timothy W. McNellis
Keyword(s):  
Fire Blight ◽  
Virulent Strain ◽  
Apple Fruit ◽  
Infection Process ◽  
Bacterial Disease ◽  
Primary Metabolism ◽  
In Planta ◽  
Coding Region ◽  
Content Type ◽  
Expression Of Genes

ABSTRACTFire blight is caused byErwinia amylovoraand is the most destructive bacterial disease of apples and pears worldwide. In this study, we found thatE. amylovoraargD(1000)::Tn5, anargDTn5transposon mutant that has the Tn5transposon inserted after nucleotide 999 in theargDgene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. TheE. amylovoraargDgene encodes a predictedN-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-typeargDgene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of theargD(1000)::Tn5mutant. However, even when mixed with virulentE. amylovoracells and inoculated onto immature apple fruit, theargD(1000)::Tn5mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argDcomplementation plasmid was stably maintained in theargD(1000)::Tn5mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argDcomplementation plasmid could be useful for the expression of genes, markers, and reporters inE. amylovoragrowingin planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered anE. amylovoravirulence factor because theargD(1000)::Tn5mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate thatE. amylovoracannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.

scholarly journals SodA Contributes to the Virulence of Avian PathogenicEscherichia coliO2 Strain E058 in Experimentally Infected Chickens

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Qingqing Gao ◽  
Le Xia ◽  
Xiaobo Wang ◽  
Zhengqin Ye ◽  
Jinbiao Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Virulence Factor ◽  
Infection Process ◽  
Strain Identification ◽  
Bacterial Disease ◽  
Wild Type ◽  
Content Type

ABSTRACTStrains of avian pathogenicEscherichia coli(APEC), the common pathogen of avian colibacillosis, encounter reactive oxygen species (ROS) during the infection process. Superoxide dismutases (SODs), acting as antioxidant factors, can protect against ROS-mediated host defenses. Our previous reports showed that thesodAgene (encoding a Mn-cofactor-containing SOD [MnSOD]) is highly expressed during the septicemic infection process of APEC.sodAhas been proven to be a virulence factor of certain pathogens, but its role in the pathogenicity of APEC has not been fully identified. In this study, we deleted thesodAgene from the virulent APEC O2 strain E058 and examined thein vitroandin vivophenotypes of the mutant. ThesodAmutant was more sensitive to hydrogen peroxide in terms of both its growth and viability than was the wild type. The ability to form a biofilm was weakened in thesodAmutant. ThesodAmutant was significantly more easily phagocytosed by chicken macrophages than was the wild-type strain. Chicken infection assays revealed significantly attenuated virulence of thesodAmutant compared with the wild type at 24 h postinfection. The virulence phenotype was restored by complementation of thesodAgene. Quantitative real-time reverse transcription-PCR revealed that the inactivation ofsodAreduced the expression of oxidative stress response geneskatE,perR, andosmCbut did not affect the expression ofsodBandsodC. Taken together, our studies indicate that SodA is important for oxidative resistance and virulence of APEC E058.IMPORTANCEAvian colibacillosis, caused by strains of avian pathogenicEscherichia coli, is a major bacterial disease of severe economic significance to the poultry industry worldwide. The virulence mechanisms of APEC are not completely understood. This study investigated the influence of an antioxidant protein, SodA, on the phenotype and pathogenicity of APEC O2 strain E058. This is the first report demonstrating that SodA plays an important role in protecting a specific APEC strain against hydrogen peroxide-induced oxidative stress and contributes to the virulence of this pathotype strain. Identification of this virulence factor will enhance our knowledge of APEC pathogenic mechanisms, which is crucial for designing successful strategies against associated infections and transmission.

scholarly journals Erwinia amylovoraAuxotrophic Mutant Exometabolomics and Virulence on Apples

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Sara M. Klee ◽  
Judith P. Sinn ◽  
Melissa Finley ◽  
Erik L. Allman ◽  
Philip B. Smith ◽  
...  
Keyword(s):  
Fire Blight ◽  
De Novo ◽  
Virulent Strain ◽  
Auxotrophic Mutant ◽  
Management Tool ◽  
Wild Type ◽  
Management Options ◽  
Substantial Impact ◽  
Content Type ◽  
Arginine Auxotroph

ABSTRACTThe Gram-negative bacteriumErwinia amylovoracauses fire blight disease of apples and pears. While the virulence systems ofE. amylovorahave been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophicE. amylovoramutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-typeE. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenicE. amylovoraauxotrophs could have utility as fire blight biocontrol agents.IMPORTANCEThis study has revealed the availability of a range of host metabolites toE. amylovoracells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterialde novosynthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophicE. amylovoramutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulentE. amylovoraon flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenicE. amylovoraauxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

scholarly journals Xylan Utilization Regulon in Xanthomonas citri pv. citri Strain 306: Gene Expression and Utilization of Oligoxylosides

2015 ◽  
Vol 81 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
V. Chow ◽  
D. Shantharaj ◽  
Y. Guo ◽  
G. Nong ◽  
G. V. Minsavage ◽  
...  
Keyword(s):  
Cell Wall ◽  
Citrus Canker ◽  
Glycoside Hydrolase Family ◽  
In Planta ◽  
Xanthomonas Citri ◽  
Plant Host ◽  
Gene Encoding ◽  
Content Type ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACTXanthomonas citripv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. InXcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, theagu67gene, encoding a GH67 α-glucuronidase (Agu67), thexyn43Egene, encoding a putative GH43 α-l-arabinofuranosidase, and thexyn43Fgene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGXn) to oligoxylosides methylglucuronoxylotriose (MeGX3), xylotriose (X3), and xylobiose (X2).Xcc306 completely utilizes MeGXnpredigested with Xyn10A or Xyn10C but shows little utilization of MeGXn.Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX3for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGXn. Preferential growth on oligoxylosides compared to growth on polymeric MeGXnindicates that GH10 xylanases, either secreted byXcc306in plantaor produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides ofxyn10,agu67,cirA, thetonBreceptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action ofXcc306 GH10 xylanases on MeGXn. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival ofXcc306 and, ultimately, to plant disease.

scholarly journals Virulence Characteristics Accounting for Fire Blight Disease Severity in Apple Trees and Seedlings

2010 ◽  
Vol 100 (6) ◽  
pp. 539-550 ◽  
Author(s):  
Steven A. Lee ◽  
Henry K. Ngugi ◽  
Noemi O. Halbrendt ◽  
Grace O'Keefe ◽  
Brian Lehman ◽  
...  
Keyword(s):  
Cell Death ◽  
Disease Severity ◽  
Biofilm Formation ◽  
Fire Blight ◽  
Apple Fruit ◽  
Bacterial Disease ◽  
Apple Trees ◽  
Carbohydrate Utilization ◽  
Hypersensitive Cell Death ◽  
Blight Disease

The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, the most destructive bacterial disease of rosaceous plants, including apple and pear. Here, we compared the virulence levels of six E. amylovora strains (Ea273, CFBP1367, Ea581a, E2002a, E4001a, and HKN06P1) on apple trees and seedlings. The strains produced a range of disease severity, with HKN06P1 producing the greatest disease severity in every assay. We then compared virulence characteristic expression among the six strains, including growth rates in immature apple fruit, amylovoran production, levansucrase activity, biofilm formation, carbohydrate utilization, hypersensitive cell death elicitation in tobacco leaves, and protein secretion profiles. Multiple regression analysis indicated that three of the virulence characteristics (amylovoran production, biofilm formation, and growth in immature apple fruit) accounted for >70% of the variation in disease severity on apple seedlings. Furthermore, in greenhouse-grown ‘Gala’ trees, >75% of the variation in disease severity was accounted for by five of the virulence characteristics: amylovoran production, biofilm formation, growth in immature apple fruit, hypersensitive cell death elicitation, and sorbitol utilization. This study demonstrates that virulence factor expression levels account for differences in disease severity caused by wild isolates of E. amylovora on apple trees.

scholarly journals Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears

2020 ◽  
Vol 367 (20) ◽  
Author(s):  
Candace R Alexander ◽  
Regan B Huntley ◽  
Neil P Schultes ◽  
George S Mourad
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Functional Characterization ◽  
Biochemical Properties ◽  
Apple Fruit ◽  
Infection Process ◽  
Disease Symptoms ◽  
Uptake Studies ◽  
Important Disease

ABSTRACT Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43 ± 0.09 μM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

scholarly journals Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

2012 ◽  
Vol 78 (16) ◽  
pp. 5638-5645 ◽  
Author(s):  
Yoon-Suk Kang ◽  
Brian Bothner ◽  
Christopher Rensing ◽  
Timothy R. McDermott
Keyword(s):  
Binding Site ◽  
Sigma Factor ◽  
Model Organism ◽  
Coding Region ◽  
Rt Pcr ◽  
Obvious Effect ◽  
Content Type ◽  
Definitive Evidence ◽  
Relative Contribution ◽  
Insertional Inactivation

ABSTRACTIn this study with the model organismAgrobacterium tumefaciens, we used a combination oflacZgene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of AsIIIoxidation. A deletion mutation that removed the RpoN binding site from theaioBApromoter and anaacC3(gentamicin resistance) cassette insertional inactivation of therpoNcoding region eliminatedaioBAexpression and AsIIIoxidation, althoughrpoNexpression was not related to cell exposure to AsIII. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters foraioB,phoB1,pstS1,dctA,glnA,glnB, andflgBthat were examined by using qualitative RT-PCR andlacZreporter fusions to assess the relative contribution of RpoN to their transcription. The expressions ofaioBanddctAin the wild-type strain were considerably enhanced in cells exposed to AsIII, and both genes were silent in therpoN::aacC3mutant regardless of AsIII. The expression level ofglnAwas not influenced by AsIIIbut was reduced (but not silent) in therpoN::aacC3mutant and further reduced in the mutant under N starvation conditions. TherpoN::aacC3mutation had no obvious effect on the expression ofglnB,pstS1,phoB1, orflgB. These experiments provide definitive evidence to document the requirement of RpoN for AsIIIoxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by AsIIIor by this sigma factor.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

scholarly journals Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect Against Citrus Huanglongbing via Trunk Injection

2016 ◽  
Vol 106 (12) ◽  
pp. 1495-1503 ◽  
Author(s):  
Jiahuai Hu ◽  
Nian Wang
Keyword(s):  
Root System ◽  
Early Stage ◽  
Spatiotemporal Dynamics ◽  
Bacterial Disease ◽  
In Planta ◽  
Control Effect ◽  
Chromatography Method ◽  
Effective Measure ◽  
Citrus Huanglongbing ◽  
Trunk Injection

Citrus huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing ‘Candidatus Liberibacter asiaticus’ (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

scholarly journals Pathogenicity and Infection Cycle of Vibrio owensii in Larviculture of the Ornate Spiny Lobster (Panulirus ornatus)

2012 ◽  
Vol 78 (8) ◽  
pp. 2841-2849 ◽  
Author(s):  
Evan F. Goulden ◽  
Michael R. Hall ◽  
David G. Bourne ◽  
Lily L. Pereg ◽  
Lone Høj
Keyword(s):  
Fluorescent Protein ◽  
Histopathological Examination ◽  
Single Cells ◽  
Larval Mortality ◽  
Spiny Lobster ◽  
Systemic Infection ◽  
Mortality Rates ◽  
Infection Process ◽  
Content Type ◽  
Live Feed

ABSTRACTThe type strain ofVibrio owensii(DY05) was isolated during an epizootic of aquaculture-reared larvae (phyllosomas) of the ornate spiny lobster (Panulirus ornatus).V. owensiiDY05 was formally demonstrated to be the etiological agent of a disease causing rapid and reproducible larval mortality with pathologies similar to those seen during disease epizootics. Vectored challenge via the aquaculture live feed organismArtemia(brine shrimp) caused consistent cumulative mortality rates of 84 to 89% after 72 h, in contrast to variable mortality rates seen after immersion challenge. Histopathological examination of vector-challenged phyllosomas revealed bacterial proliferation in the midgut gland (hepatopancreas) concomitant with epithelial cell necrosis. A fluorescent-protein-labeledV. owensiiDY05 transconjugant showed dispersal of single cells in the foregut and hepatopancreas 6 h postexposure, leading to colonization of the entire hepatopancreas within 18 h and eventually systemic infection.V. owensiiDY05 is a marine enteropathogen highly virulent toP. ornatusphyllosoma that uses vector-mediated transmission and release from host association to a planktonic existence to perpetuate transfer. This understanding of the infection process will improve targeted biocontrol strategies and enhance the prospects of commercially viable larviculture for this valuable spiny lobster species.

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