scholarly journals Exploring the pathogenic function of an endogenous plasmid of Pantoea ananatis by a simple and efficient plasmid elimination strategy

2019 ◽  
Author(s):  
Xiaozhen Zhao ◽  
Lu Gao ◽  
Hai Huang ◽  
Yi Zhao ◽  
Alvina Hanif ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Plasmid Dna ◽  
Brown Rot ◽  
Economic Losses ◽  
Temperature Sensitive ◽  
Pantoea Ananatis ◽  
Virulence Plasmids ◽  
Maize Plants ◽  
Plasmid Elimination

Abstract Background: The bacterium Pantoea ananatis is associated with devastating diseases in many crops that cause serious economic losses. We previously isolated strain DZ-12 from maize brown rot leaves and, genome sequencing revealed that it belongs to P. ananatis and contains a large, endogenous plasmid, pDZ-12. Virulence plasmids are essential for pathogenesis in many bacterial pathogens. However, nothing was known regarding the role of this plasmid in P. ananatis pathogenicity in maize. Results: Here, we eliminated the endogenous plasmid from P. ananatis by substituting its native replicon with a temperature-sensitive replicon. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37 °C). Loss of pDZ-12 from P. ananatis DZ-12 led to decreased disease severity in maize plants, suggesting the endogenous plasmid was important for pathogenesis. Meanwhile, loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The method described here, which is efficient and needs only two steps to cure the endogenous plasmid without antibiotic resistance, was also shown to work in Bacillus subtilis, and may be generally applicable in bacteria. Conclusions: This study provides the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and in the ability of this species to form biofilms. It also presents the first report on curing plasmid DNA from P. ananatis.

scholarly journals Exploring the pathogenic function of endogenous plasmid from Pantoea ananatis by a simple and efficient plasmid elimination strategy

2019 ◽  
Author(s):  
Xiaozhen Zhao ◽  
Lu Gao ◽  
Hai Huang ◽  
Yi Zhao ◽  
Alvina Hanif ◽  
...  
Keyword(s):  
Brown Rot ◽  
Economic Losses ◽  
Temperature Sensitive ◽  
Pantoea Ananatis ◽  
Virulence Plasmids ◽  
Plasmid Replicon ◽  
Wide Range ◽  
Bacterium Bacillus Subtilis ◽  
Maize Plants ◽  
Bacterium Bacillus

Abstract Background: Pantoea ananatis, a Gram-negative bacterium and species of the family Enterobacteriaceae, is associated with diseases in a wide range of crops. Diseases caused by P. ananatis are devastating and can result in serious economic losses. We previously isolated strain DZ-12 from maize brown rot leaves and, genome sequencing revealed that it belongs to P. ananatis and contains a large, endogenous plasmid, pDZ-12. Virulence plasmids are essential for pathogenesis in many bacterial pathogens. However, nothing was known regarding the role of this plasmid in the pathogenicity of P. ananatis in maize plants. Results: Here, we developed a simple and efficient strategy to eliminate the endogenous plasmid by substituting its native replicon with a temperature-sensitive replicon. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37 ℃). Loss of pDZ-12 from P. ananatis DZ-12 led to decreased disease severity in maize plants, suggesting the endogenous plasmid was important for pathogenesis. Meanwhile, loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The method described here, which is efficient and needs only two steps to cure the endogenous plasmid without antibiotic resistance, was also shown to work in the Gram-positive bacterium Bacillus subtilis, and may be generally applicable in bacteria. Conclusions: This study we provide the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and in the ability of this species to form biofilms, which further our understanding of the pathogenicity of P. ananatis. It also presents the first report on curing plasmid DNA from P. ananatis. Keywords: Pantoea ananatis, endogenous plasmid, replicon, temperature-sensitive

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Relationship between the Tsh Autotransporter and Pathogenicity of Avian Escherichia coli and Localization and Analysis of the tsh Genetic Region

2000 ◽  
Vol 68 (7) ◽  
pp. 4145-4154 ◽  
Author(s):  
Charles M. Dozois ◽  
Maryvonne Dho-Moulin ◽  
Annie Brée ◽  
John M. Fairbrother ◽  
Clarisse Desautels ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Virulence Plasmid ◽  
Temperature Sensitive ◽  
E Coli ◽  
Virulence Plasmids ◽  
V Genes ◽  
Avian Origin ◽  
Colicin V

ABSTRACT The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain χ7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among thetsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with χ7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain χ7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain χ7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of theEnterobacteriaceae. These results demonstrate that thetsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

scholarly journals Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Selene Rubiola ◽  
Tiziana Civera ◽  
Felice Panebianco ◽  
Davide Vercellino ◽  
Francesco Chiesa
Keyword(s):  
18S Rdna ◽  
Inflammatory Myopathy ◽  
Molecular Techniques ◽  
Diaphragm Muscle ◽  
Economic Losses ◽  
Intermediate Hosts ◽  
Slaughter Cattle ◽  
Significant Difference ◽  
Pcr Targeting

Abstract Background Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. Methods To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. Results Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. Conclusions Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis. Graphic Abstract

scholarly journals DNA repair and the evolution of transformation in Bacillus subtilis. II. Role of inducible repair.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 411-422
Author(s):  
M F Wojciechowski ◽  
M A Hoelzer ◽  
R E Michod
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Physiological State ◽  
Transformation Rate ◽  
Exogenous Dna ◽  
Expression Of Genes ◽  
Cell Subpopulations ◽  
Experimental Treatments

Abstract In Bacillus subtilis, DNA repair and recombination are intimately associated with competence, the physiological state in which the bacterium can bind, take up and recombine exogenous DNA. Previously, we have shown that the homologous DNA transformation rate (ratio of transformants to total cells) increases with increasing UV dosage if cells are transformed after exposure to UV radiation (UV-DNA), whereas the transformation rate decreases if cells are transformed before exposure to UV (DNA-UV). In this report, by using different DNA repair-deficient mutants, we show that the greater increase in transformation rate in UV-DNA experiments than in DNA-UV experiments does not depend upon excision repair or inducible SOS-like repair, although certain quantitative aspects of the response do depend upon these repair systems. We also show that there is no increase in the transformation rate in a UV-DNA experiment when repair and recombination proficient cells are transformed with nonhomologous plasmid DNA, although the results in a DNA-UV experiment are essentially unchanged by using plasmid DNA. We have used din operon fusions as a sensitive means of assaying for the expression of genes under the control of the SOS-like regulon in both competent and noncompetent cell subpopulations as a consequence of competence development and our subsequent experimental treatments. Results indicate that the SOS-like system is induced in both competent and noncompetent subpopulations in our treatments and so should not be a major factor in the differential response in transformation rate observed in UV-DNA and DNA-UV treatments. These results provide further support to the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in the repair of DNA damage.

scholarly journals Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation

2021 ◽  
Vol 9 (5) ◽  
pp. 1046
Author(s):  
Inam Ul Haq ◽  
Sabine Brantl
Keyword(s):  
Bacillus Subtilis ◽  
Antisense Rna ◽  
Rna Binding ◽  
Rna Degradation ◽  
Metabolic Enzyme ◽  
Dual Function ◽  
Small Proteins ◽  
Moonlighting Proteins ◽  
Rnase Y

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

scholarly journals Analysis of the Role of Bradysiaimpatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1546
Author(s):  
Marta Budziszewska ◽  
Patryk Frąckowiak ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Nicotiana Benthamiana ◽  
Molecular Mechanisms ◽  
Virus Transmission ◽  
Pathogenic Fungi ◽  
Economic Losses ◽  
Fungus Gnats ◽  
Droplet Pcr ◽  
Viral Coat ◽  
Peanut Stunt Virus

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

scholarly journals Immunogenicity and protective efficacy of enterotoxigenic Escherichia coli (ETEC) total RNA against ETEC challenge in a mouse model

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mandi Liu ◽  
Yue Zhang ◽  
Di Zhang ◽  
Yun Bai ◽  
Guomei Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mouse Model ◽  
Immune Responses ◽  
Protective Efficacy ◽  
Enterotoxigenic Escherichia Coli ◽  
Economic Losses ◽  
Th2 Response ◽  
Total Rna ◽  
Etec Infection

AbstractEnterotoxigenic Escherichia coli (ETEC), an essential cause of post-weaning diarrhea (PWD) in piglets, leads to significant economic losses to the pig industry. The present study aims to identify the role of ETEC total RNA in eliciting immune responses to protect animals against ETEC infection. The results showed that the total RNA isolated from pig-derived ETEC K88ac strain effectively stimulated the IL-1β secretion of porcine intestinal epithelial cells (IPEC-J2). The mouse model immunized with ETEC total RNA via intramuscular injection (IM) or oral route (OR) was used to evaluate the protective efficiency of the ETEC total RNA. The results suggested that 70 μg ETEC total RNA administered by either route significantly promoted the production of the serum IL-1β and K88ac specific immunoglobulins (IgG, IgM, and IgA). Besides, the ETEC RNA administration augmented strong mucosal immunity by elevating K88ac specific IgA level in the intestinal fluid. Intramuscularly administered RNA induced a Th1/Th2 shift toward a Th2 response, while the orally administered RNA did not. The ETEC total RNA efficiently protected the animals against the ETEC challenge either by itself or as an adjuvant. The histology characterization of the small intestines also suggested the ETEC RNA administration protected the small intestinal structure against the ETEC infection. Particularly of note was that the immunity level and protective efficacy caused by ETEC RNA were dose-dependent. These findings will help understand the role of bacterial RNA in eliciting immune responses, and benefit the development of RNA-based vaccines or adjuvants.

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