Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression

2004 ◽  
Vol 50 (7) ◽  
pp. 509-513 ◽  
Author(s):  
Jayaraman Jayaraj ◽  
Subbaratnam Muthukrishnan ◽  
George H Liang
Keyword(s):  
Azospirillum Brasilense ◽  
Pathogenic Fungus ◽  
Chitinase Activity ◽  
Chitinase Gene ◽  
Cell Protein ◽  
Broad Host Range ◽  
Nitrogen Fixing ◽  
Rice Chitinase ◽  
Chitinase Genes

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35–40 × 10–6. The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.Key words: Azospirillum-transformation, rice chitinase gene, protein expression, chitinase activity.

scholarly journals Enhanced Resistance To Fungal Pathogens in Transgenic Peanut (Arachis Hypogaea L.) Cultivar L14 by Overexpression of Gene encoding Chitinase 42 kDa from Trichoderma Asperellum SH16

2021 ◽  
Author(s):  
Phung Thi Bich Hoa ◽  
Nguyen Hoang Tue ◽  
Huynh Thi Quynh Trang ◽  
Hoang Anh Thu ◽  
Le Ngoc Huyen Nhung ◽  
...  
Keyword(s):  
Arachis Hypogaea ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Chitinase Activity ◽  
Trichoderma Asperellum ◽  
Wild Type ◽  
Synthetic Genes ◽  
Chitinase Genes ◽  
Transgenic Peanut

Abstract This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specific Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the field of transgenic crops against phytopathogenic fungi.

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 921-928 ◽  
Author(s):  
Serena Selvaggini ◽  
Carol A. Munro ◽  
Serge Paschoud ◽  
Dominique Sanglard ◽  
Neil A. R. Gow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
De Novo ◽  
Gene Families ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Growing Cell ◽  
Chitinase Genes

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

scholarly journals Knockout of the Rodent Malaria Parasite Chitinase PbCHT1 Reduces Infectivity to Mosquitoes

2001 ◽  
Vol 69 (6) ◽  
pp. 4041-4047 ◽  
Author(s):  
Johannes T. Dessens ◽  
Jacqui Mendoza ◽  
Charles Claudianos ◽  
Joseph M. Vinetz ◽  
Emad Khater ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Malaria Parasite ◽  
Avian Malaria ◽  
Chitinase Activity ◽  
Chitinase Gene ◽  
Rodent Malaria Parasite ◽  
Human Malaria Parasite ◽  
Rodent Malaria ◽  
Human Malaria

ABSTRACT During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite,Plasmodium berghei. We show that the gene, namedPbCHT1, is a structural ortholog ofPgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity inAnopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds—an artificial situation where midgut invasion occurs before PM formation—suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution ofPlasmodium-mosquito interactions.

scholarly journals Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Adamczyk ◽  
E. Lewicka ◽  
R. Szatkowska ◽  
H. Nieznanska ◽  
J. Ludwiczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Broad Host Range ◽  
Biophysical Properties ◽  
Dna Binding Sites ◽  
In Silico Studies ◽  
Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Identifying Phenotypically Distinct Fibroblast Subsets in Type 2 Diabetes–Associated Iatrogenic Laryngotracheal Stenosis

2021 ◽  
pp. 019459982110147
Author(s):  
Ioan A. Lina ◽  
Alexandra Berges ◽  
Rafael Ospino ◽  
Ruth J. Davis ◽  
Kevin M. Motz ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Flow Cytometry ◽  
Pearson Correlation ◽  
Phenotypic Expression ◽  
In Vitro Study ◽  
Cell Protein ◽  
Laryngotracheal Stenosis ◽  
Tertiary Referral Center

Objective Iatrogenic laryngotracheal stenosis (iLTS) is the pathologic narrowing of the glottis, subglottis, and/or trachea secondary to intubation or tracheostomy related injury. Patients with type 2 diabetes mellitus (T2DM) are more likely to develop iLTS. To date, the metabolomics and phenotypic expression of cell markers in fibroblasts derived from patients with T2DM and iLTS are largely unknown. Study Design Controlled in vitro cohort study. Setting Tertiary referral center (2017-2020). Methods This in vitro study assessed samples from 6 patients with iLTS who underwent surgery at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea and compared with controls obtained from the trachea of rapid autopsy specimens. Patients with iLTS were subcategorized into those with and without T2DM. Metabolic substrates were identified by mass spectrometry, and cell protein expression was measured by flow cytometry. Results T2DM iLTS-scar fibroblasts had a metabolically distinct profile and clustered tightly on a Pearson correlation heat map as compared with non-T2DM iLTS-scar fibroblasts. Levels of itaconate were elevated in T2DM iLTS-scar fibroblasts. Flow cytometry demonstrated that T2DM iLTS-scar fibroblasts were associated with higher CD90 expression (Thy-1; mean, 95%) when compared with non-T2DM iLTS-scar (mean, 83.6%; P = .0109) or normal tracheal fibroblasts (mean, 81.1%; P = .0042). Conclusions Scar-derived fibroblasts from patients with T2DM and iLTS have a metabolically distinct profile. These fibroblasts are characterized by an increase in itaconate, a metabolite related to immune-induced scar remodeling, and can be identified by elevated expression of CD90 (Thy-1) in vitro.

scholarly journals Peyer's Patch Dendritic Cells Process Viral Antigen from Apoptotic Epithelial Cells in the Intestine of Reovirus-infected Mice

2004 ◽  
Vol 200 (2) ◽  
pp. 235-245 ◽  
Author(s):  
Marina N. Fleeton ◽  
Nikhat Contractor ◽  
Francisco Leon ◽  
J. Denise Wetzel ◽  
Terence S. Dermody ◽  
...  
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Cd4 T Cells ◽  
Cell Protein ◽  
Peyer’S Patch ◽  
Peyer's Patch ◽  
Antigen Uptake ◽  
Cross Presentation

We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (σ1) and nonstructural (σNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both σ1 and σNS, indicating productive viral replication. In contrast, σ1, but not σNS, was detected in the subepithelial dome (SED) in association with CD11c+/CD8α−/CD11blo DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained σ1, but not σNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, σ1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4+ T cells in vitro. These studies show that CD8α−/CD11blo DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4+ T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments

1995 ◽  
Vol 15 (1) ◽  
pp. 61-71 ◽  
Author(s):  
P H Watson ◽  
S T Mortimer ◽  
K K W Wang ◽  
D E Croall ◽  
D A Hanley
Keyword(s):  
Parathyroid Hormone ◽  
Parathyroid Tissue ◽  
Area Under The Curve ◽  
Molar Ratio ◽  
Cell Protein ◽  
Parathyroid Cell ◽  
Peptide Fragments ◽  
Intracellular Degradation ◽  
Peak Areas

ABSTRACT Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca2+-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca2+-regulated PTH secretion. The present study was undertaken to determine the presence of calciumactivated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71·5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47–84 and bPTH69–84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments.

scholarly journals Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2904-2911 ◽  
Author(s):  
Michael Wyrebek ◽  
Cristina Huber ◽  
Ramanpreet Kaur Sasan ◽  
Michael J. Bidochka
Keyword(s):  
Tree Species ◽  
Acer Saccharum ◽  
Panicum Virgatum ◽  
Sugar Maple ◽  
Root Exudate ◽  
Pathogenic Fungus ◽  
Plant Roots ◽  
Metarhizium Robertsii ◽  
Grass Roots

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5′ EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.

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