scholarly journals Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex

2017 ◽  
Vol 30 (7) ◽  
pp. 589-600 ◽  
Author(s):  
Prem P. Kandel ◽  
Rodrigo P. P. Almeida ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Homologous Recombination ◽  
Fluorescent Protein ◽  
Xylella Fastidiosa ◽  
Dna Recombination ◽  
Bacterial Cells ◽  
Disease Emergence ◽  
Natural Competence ◽  
In Silico Studies ◽  
Green Fluorescent

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

scholarly journals Obtaining a fluorescently labeled endophytic strain of bacteria Herbaspirillum lusitanum P6-12 for their detection in vivo and in vitro

2021 ◽  
Vol 21 (3) ◽  
pp. 286-291
Author(s):  
Arapat Rustamovna Bagavova ◽  
◽  
Natal’ya S. Velichko ◽  
Timofey E. Pylayev ◽  
Yuliya P. Fedonenko ◽  
...  
Keyword(s):  
Recombinant Strain ◽  
Fluorescent Protein ◽  
Biochemical Properties ◽  
Growth Media ◽  
Bacterial Cells ◽  
Vector Plasmid ◽  
Green Fluorescent ◽  
Model Strain

The Herbaspirillum lusitanum P6-12 strain containing the vector plasmid pJN105TurboGFP, which encodes the synthesis of the green fluorescent protein GFP, and which has resistance to the antibiotic gentamicin, was obtained by electroporation. The constructed strain of H. lusitanum P6-12 in cultural, morphological and biochemical properties did not differ from the original typical natural strain of H. lusitanum P6-12. On solid growth media, the recombinant strain formed yellow-green colonies, fluorescent under UV irradiation. Upon inoculation with the resulting culture of plant objects, a green glow of the marked H. lusitanum P6-12 cells, actively colonizing the internal tissues of the host plant, was observed. The created strain can be used as a model strain for studying the patterns and characteristics of the behaviour of organisms in integrated systems, including for tracking bacterial cells during interaction with plants, assessing their survival, competitiveness, etc.

The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

scholarly journals Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be “Flying Syringes”

2015 ◽  
Vol 105 (5) ◽  
pp. 608-620 ◽  
Author(s):  
Elaine A. Backus ◽  
Holly J. Shugart ◽  
Elizabeth E. Rogers ◽  
J. Kent Morgan ◽  
Robert Shatters
Keyword(s):  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Xylella Fastidiosa ◽  
Conclusive Evidence ◽  
Fluorescent Nanoparticles ◽  
Bacterial Cells ◽  
Homalodisca Vitripennis ◽  
Artificial Diets ◽  
New Paradigm ◽  
Green Fluorescent

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector’s stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector’s transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true “flying syringes,” especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

scholarly journals Expression of Duplicate msa Genes in the Salmonid Pathogen Renibacteriumsalmoninarum

2002 ◽  
Vol 68 (11) ◽  
pp. 5480-5487 ◽  
Author(s):  
Linda D. Rhodes ◽  
Alison M. Coady ◽  
Mark S. Strom
Keyword(s):  
Homologous Recombination ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Soluble Antigen ◽  
Positive Bacterium ◽  
Renibacterium Salmoninarum ◽  
Gene Encoding ◽  
Bacterial Kidney Disease ◽  
Green Fluorescent

ABSTRACT Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum.

Enhancement of population densities of fluorescent pseudomonads in the rhizosphere of tomato plants by addition of acibenzolar-S-methyl

2002 ◽  
Vol 48 (12) ◽  
pp. 1069-1075 ◽  
Author(s):  
W D Fakhouri ◽  
H Buchenauer
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Pseudomonads ◽  
Fluorescent Pseudomonad ◽  
Bacterial Cells ◽  
In Vitro Production ◽  
Tomato Plants ◽  
Population Sizes ◽  
Cell Densities ◽  
Green Fluorescent

Fluorescent pseudomonad isolates G309 and CW2, in combination with the resistance inducer acibenzolar-S-methyl (ASM), improved control of fungal and bacterial diseases on tomato plants. The interactions of the bacteria in the presence of ASM showed that in vitro growth of Pseudomonas fluorescens G309 and Pseudomonas sp. strain CW2 was not affected in King's B broth supplemented with 10 and 20 μM ASM. Also, the bacterial cells were not able to utilize ASM as a nutrient source. In vitro production of the two antimicrobial secondary metabolites phenazine-1-carboxylic acid and 2-OH-phenazine by the isolate CW2 was not affected within 3 days from incubation. In contrary, addition of ASM at a concentration of 20 μM to King's B liquid medium significantly increased production of salicylic acid by isolate G309. When roots of tomato plants were treated with G309 or CW2 cell suspensions containing 20 μM ASM, the number of bacterial cells recovered from the rhizosphere was significantly higher in the combined treatments than in the single applications 5, 10, and 15 days after inoculation. However, ASM at a higher concentration (50 μM) did not appreciably enhance the population sizes of either bacterial isolate in the rhizosphere. Enhanced bacterial cell densities in the rhizosphere of tomato plants were also determined following simultaneous treatments of tomato roots with 10 and 20 μM ASM in combination with the transformed isolate G309-384 (mini-Tn5gfp), which encodes the green fluorescent protein.Key words: acibenzolar-S-methyl, fluorescent pseudomonads, green fluorescent protein, tomato.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andi R. Sultan ◽  
Kirby R. Lattwein ◽  
Nicole A. Lemmens-den Toom ◽  
Susan V. Snijders ◽  
Klazina Kooiman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Clonal Complex ◽  
Regulatory Pathways ◽  
Immune Modulator ◽  
Viability Assay ◽  
Analgesic Agents ◽  
Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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