Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.

Pectinolytic enzymes [pectinases] produced by microbes are highly important for their biotechnological use in processing of vegetables and fruits beverages and use in pulp and paper industry. A pectinases, namely endo-polygalacturonase [endo-PGase], encoding gene isolated from Pectobacterium carotovorum, a plant pathogenic strain of bacteria was successfully cloned into a secretion vector pHT43 having σ?-dependent promoter P grac . For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium. The recombinant B. subtilis competent cells, harboring the engineered pHT43 with the endo-PGase gene were cultured in 2X-yeast extract tryptone medium. The recombinant endo-PGase enzyme was secreted directly into the medium after 72 hours of the first IPTG induction. The recombinant endo-PGase was screened for its activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40°C with a stability ranging from pH 5.0-9.0. For detection of metal ion effect, recombinant enzyme was incubated with 1mM concentration of; Ca ++ , Mg ++ , Zn ++ , EDTA, K ++ for 45 minutes. Resultantly, Ca ++ , EDTA and Zn ++ strongly inhibited the enzyme activity. The chromatographic analysis of enzymatic hydrolysate of polygalacturonic acid [PGA] and pectin substrates using HPLC and TLC revealed that tri and tetra-galacturonates were the end products of hydrolysis. The study led to the conclusion that endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi to escape endotoxins.

Why did the COVID-19 Epidemic Stop in China and does not Stop in the Rest of the World? (Application of the Two-Component Model)

The vastly different courses of the COVID-19 epidemic in China and the rest of the world are investigated and explained within two-component epidemic model. The model is based on separate accounting for the contribution to the epidemic from two types of immune response to a viral infection - innate and adaptive immunity. Any infected person becomes asymptomatic with probability (1−𝑝) or symptomatic with probability 𝑝. In the first case, innate immunity is sufficient to protect a person. In the second case, innate immunity is insufficient, and adaptive immunity comes into play. In the asymptomatic state, the person remains outwardly healthy, mobile and can spread the infection. In the symptomatic state, the person becomes ill, isolated and cannot spread the infection. We assume that the contribution to the epidemic process from asymptomatic carriers is dominant in comparison with the contribution from the usual incubation period in the symptomatic state. The key parameters of the model are the virus lifetime 𝑇 in the asymptomatic state and the spread rate 𝛽. At moderate 𝛽𝑇 values, the model describes a long, slowly decreasing morbidity plateau, which transforms into wave-like solution at 𝛽𝑇. In the case of 𝛽𝑇→∞, which corresponds to a stable non-pathogenic strain, the model solution is limited to single wave only. We believe that the spread of such a non-pathogenic strain and its subsequent dominance is responsible for ending the epidemic after the single wave of incidence in China. A way to stop the epidemic in the rest of the world may consist in displacing the circulating pathogenic virus with its stable non-pathogenic strain. Doi: 10.28991/SciMedJ-2021-0302-2 Full Text: PDF

Unraveling the Mode of Action of Cordyceps fumosorosea: Potential Biocontrol Agent against Plutella xylostella (Lepidoptera: Plutellidae)

The entomopathogenic fungus, Cordyceps fumosorosea is a potential eco-friendly biocontrol agent. The present study revealed the entire course of infection of P. xylostella by C. fumosorosea with particular reference to cuticular penetration. Comparative studies on the infection of Plutella xylostella larvae by two strains of C. fumosorosea with different pathogenicity were carried out using light, scanning, and transmission electron microscopy. We found that C. fumosorosea tended to adhere to the cuticle surfaces containing protrusions. Although conidia of the lower pathogenic strain IFCF-D58 germinated, they failed to penetrate and complete the development cycle. In contrast, the higher pathogenic strain IFCF01 began to germinate within 4 h and attached to the cuticle by a thin mucilaginous matrix within 8 h post-inoculation. After 24 h post-inoculation, germ tubes and penetrating hyphae reached the cuticular epidermis and began to enter the haemocoel. Within 36 h post-inoculation, the hyphal bodies colonized the body cavity. Hyphae penetrated from inside to outside of the body after 48 h and sporulated the cadavers. After 72 h post-inoculation, numerous conidia emerged and the mycelial covered the entire cuticular surface. The two strains showed similarities in terms of conidial size and germination rate. However, IFCF-D58 exhibited significantly fewer appressoria and longer penetrating hyphae compared to the more infective IFCF01 on all surface topographies. The current pathogen invasion sequence of events suggested that the aggressive growth and propagation along with rapid and massive in vivo production of blastospores facilitate the conidia of IFCF01 to quickly overcome the diamondback moth’s defense mechanism.

Improved De Novo Draft Genome Sequence of the Nocavionin-Producing Type Strain Nocardia terpenica IFM 0706 and Comparative Genomics with the Closely Related Strain Nocardia terpenica IFM 0406

ABSTRACT We report an improved de novo draft genome sequence of the human-pathogenic strain Nocardia terpenica IFM 0706T. The resequencing unveiled that the genome size is larger than anticipated, reducing significantly the number of contigs and building a basis for comparison with the closely related strain N. terpenica IFM 0406.