Pink Strain Research Articles

High yield expression and purification of Aspergillus flavus uricase cloned in Pichia pink™ expression system.

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60h, the fermentation process produced 1500g of biomass from 4L of semi defined culture media and expressed 2.5g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study.

Antibacterial activity of endophytes from Musa Paradisiaca

This study was aimed to isolate the endophytic bacteria and fungi from the fruit and root of Musa Paradisiaca, commonly called as Nendran banana cultivar in the southern Indian state of Kerala. Endophytes are microbes living in plant tissue without harming the plants rather existing as potential beneficial organisms. Musa paradisiaca is a less explored variety of banana cultivar which possess anti-disease potential apart from nutritional quality. The total number of twelve root and fruit associated endophytes were isolated. The fruit associated endophytes were seven with four bacterial isolates and three fungal isolates. The number of root-associated endophytes were five with three bacterial isolates and two fungal isolates. The morphological characterisation of both root and fruit endophytes were performed by Gram staining for bacteria and lacto-phenol cotton blue staining for fungi. The bacterial colony examination of the fruit associated culture revealed a specific pink strain with an inhibitory zone proving antibacterial activity which was further investigated through agar well diffusion test. The strain exhibited potential antibacterial activity against five human clinical pathogens. A significant zone of inhibition was observed against Bacillus subtilis (22 mm), Escherichia coli (21 mm), Klebsiella pneumonia and Pseudomonas aeruginosa (18 mm) which confirmed the potential antibacterial property of the fruit endophytic strain.

Shared and Independent Genetic Basis of Resistance to Bt Toxin Cry2Ab in Two Strains of Pink Bollworm

Evolution of pest resistance threatens the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Field populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton, have evolved practical resistance to transgenic cotton producing Bt toxin Cry2Ab in India, but not in the United States. Previous results show that recessive mutations disrupting an autosomal ATP-binding cassette gene (PgABCA2) are associated with pink bollworm resistance to Cry2Ab in field-selected populations from India and in one lab-selected strain from the United States (Bt4-R2). Here we discovered that an independently derived, lab-selected Cry2Ab-resistant pink bollworm strain from the United States (BX-R) also harbors mutations that disrupt PgABCA2. Premature stop codons introduced by mis-splicing of PgABCA2 pre-mRNA were prevalent in field-selected larvae from India and in both lab-selected strains. The most common mutation in field-selected larvae from India was also detected in both lab-selected strains. Results from interstrain crosses indicate BX-R has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. We conclude that recessive mutations disrupting PgABCA2 are the primary, but not the only, mechanism of resistance to Cry2Ab in pink bollworm.

Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system

l-Asparaginase is an anti-cancer agent which prevents the proliferation of cancerous cells by decreasing the level of asparagine in the blood. l-asparaginase from Escherichia coli which is encoded by ansB gene is widely used because of its substrate specificity and less glutaminase activity. Here, we are reporting the expression studies in yeast which has many added advantages like protein folding and processing. The expression studies were carried out in a new protein expression system based on the yeast Pichia pastoris called PichiaPink™. ansB gene isolated from E. coli which is coded for asparaginase was cloned into pPink HC-α plasmid and transformed into protease knock out pichia pink strain by electroporation. The recombinant enzyme was extracellular and showing the activity of 2.5IU/ml. It was then purified using Ni-NTA column since the enzyme contains His-tag at the C-terminal end. The new way of expression would be efficient in making the enzyme humanized by glycosylation patterns which is similar to mammals.

A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.

Characterization of bacteria isolated from maize roots: Emphasis on Serratia and infestation with corn rootworms (Chrysomelidae: Diabrotica)

Larval corn rootworms (Coleoptera: Chrysomelidae) are soil-dwelling insect pests that damage maize ( Zea mays L.) by consuming root tissues, thus lowering grain yield. Little is known about interactions between rootworms and soil bacteria, including potential impacts of maize rhizobacteria, such as entomopathogenic Serratia spp., on subterranean rootworm pests. We used selective growth medium (caprylate-thallous agar, CT) to quantify and isolate Serratia spp. from: (1) bulk soil, (2) roots of four field-grown maize genotypes, half of which were infested with rootworm eggs from a reared colony, and (3) non-diseased, larval rootworms from the same colony. Phenotypic testing and 16S rRNA gene sequencing were used to identify bacteria, including non- Serratia spp., that were successfully isolated on the CT solid medium. We also isolated and identified Serratia spp. associated with non-diseased and diseased Diabrotica adults. Serratia spp. associated with maize roots were more abundant than those associated with bulk soil, where they were undetectable with our methods except for Serratia grimesii. There was no impact of plant genotype on densities of bacteria isolated from maize roots. S. grimesii was frequently associated with maize roots, regardless of infestation with rootworm eggs. Serratia marcescens biotype A4, Serratia plymuthica and several other Enterobacteriaceae genera were also associated with maize roots. Infesting the soil with rootworm eggs enhance the occurrence of two strains of the S. marcescens biotype A1b, with comparable densities of these orange and pink strains within infested roots. However, both strains were associated with larval rootworms from the reared colony, which may indicate that rootworms were introducing these bacteria into infested maize roots. In addition, within larvae the orange strain was significantly more abundant than the pink strain, and was also associated with diseased rootworm adults. Our studies identified specific Serratia strains associated with diseased rootworms that may have potential as biological control agents, and additional Serratia biotypes associated with the maize rhizosphere that, based on the literature, may function as plant growth promoting agents via antagonistic action against plant-pathogenic fungi.

CAROTENE AND CYTOCHROME c DEPENDENT MITOTIC RECOMBINATION AND KILLING IN RESPONSE TO VISIBLE LIGHT AND UV‐C IN THE SMUT FUNGUS Ustilago Violacea

Abstract— The carotene (carotenoid hydrocarbons) and cytochrome c compositions for the phlph+ pink diploid strain of Ustilago violacea and three newly developed color variants, white (w), orange (o), and yellow (y) were quantitatively determined using high‐performance liquid chromatography of lipid extracts and difference spectroscopy of alkali extracts. In addition, the effect of high‐intensity incandescent and far UV (UV‐C) radiation on survival and mitotic recombination in all four phlph+ strains were studied. The phlph+w and phlph+y strains contained relatively small amounts of cytochrome c; however, the phlph+y strain accumulated β‐carotene while in the phlph+w strain only the colorless carotene phytoene was found. The phlph+w was very sensitive to incandescent radiation, with complete killing by 90 min of exposure. The induction of mitotic recombination in the phlph+w strain was inversely proportional to the level of survival, with 96% induction by 90 min of exposure. The β‐carotene‐accumulating phlph+y strain was considerably more resistant to photokilling, and exhibited induction of mitotic recombination at a lower level. Similar results were observed with these strains in response to UV‐C. The phlph+y strain was significantly more resistant to UV‐C killing than the phlph+w strain and it also exhibited lower induced levels of mitotic recombination.The phlph+o and phlph+ pink strains accumulated over 10 times the cytochrome c as the phlph+w or phlph+y strains. The phlph+o strain accumulated β‐carotene at the level of the phlph* y strain, but the phlph+ pink strain contained only about one‐tenth as much β‐carotene. The phlph+ o and phi ph+ pink strains exhibited sensitivity to visible radiation that was intermediate to the phlph+y and phlph+w strains. Mitotic recombination induction by visible radiation in the orange and pink strains was slightly less than that in the phlph+w strain. In response to UV‐C, the phlph+o and phlph+ pink strains had survival and mitotic recombination induction characteristics that were similar to the phlph+w strain.

Pigmentation of Ustilago scabiosae by carotene and cytochrome caccumulation

Four phenotypically yellow, pink, and orange haploid strains of Ustilago scabiosae were analyzed for carotene composition using high-performance liquid chromatography of lipid extracts. Cytochromec concentration was also determined by difference spectrophotometry of cell extracts. The yellow phenotypes resulted primarily from the yellow carotenes,β-carotene andcis-β-zeacarotene, while the main carotene in the orange strain was lycopene, in addition toγ-carotene, neurosporene, andβ-carotene. The pink strain contained lycopene,γ-carotene,cis-β-zeacarotene, andβ-carotene, as well as about twice the amount of cytochromec as the other strains. All of the tested strains also contained the colorless carotene phytoene.

Photosensitivity of pigmented and nonpigmented strains ofUstilago violacea

White, pink, pumpkin, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities in the presence and absence of the endogenous photosensitizer toluidine blue (TB). The white strain 15.10 and the cytochrome-c-accumulating pink strain 1.C429 were completely lacking carotenes and were the most sensitive to visible light under all conditions. The phytoene-containing white strain 2.C419, the carotene-containing pink strains AB278a-1, 1.C421, and 2.C425, and pumpkin strain 2.C415 were more resistant to photodestruction in the presence and absence of TB than strains 15.10 and 1.C429. Consistently, the most light resistant strains were the high carotene, low cytochrome-c-accumulating orange, and yellow strains. The photosensitivity of strains ofU. violacea was related to the level of carotene, cytochrome c, the presence or absence of TB, and the age of the culture.

Characterization of the pigment in pink sporidial colonies of Ustilago violacea as cytochrome c

The pigment of a pink strain of Ustilago violacea 1.C429 was associated with the mitochondria isolated on a sucrose gradient. The extracted red pigment was identified as cytochrome c by spectral analysis and by oxidation with cytochrome c oxidase from rat liver mitochondria. The pink strain accumulated at least tenfold more cytochrome c than a white strain. Zn 2+ enhanced cytochrome c production in a white strain by 75%.

An intrinsic factor influencing alate production by two strains of the pea aphid, Acyrthosiphon pisum

Alate virginoparae of a pink and a green strain of pea aphid and both first-born and last-born representatives of several successive generations of their apterous descendants were exposed to a standard crowding treatment in order to determine whether or not they could be induced to give birth to alate offspring. Alatae of the pink strain which were crowded shortly after the onset of parturition subsequently produced very few alate progeny. Their first-born apterous daughters, however, when similarly tested were highly responsive to this stimulus as were all apterae of the next two generations. Crowded alatae of the green strain produced no alate offspring. The ability to respond to such tactile stimulation returned after a period of time to some of their descendants, was lost in females born later, but was found still later to return again. The existence of some sort of timing mechanism is suggested by the present results since representatives of various generations of this strain regained simulataneously and later lost the ability to respond to a crowding stimulus.

Studies on the pink, adenine-deficient strains ofCandida albicans

Several pink or dark brown mutants of Candida albicans were obtained following exposure of the wild types to ultraviolet irradiation or ethyl methanesulfonate. Unlike the wild types, all the mutants required adenine and in addition the dark brown strain required valine and isoleucine for growth. The mutants accumulated the pigment in the cytoplasm either as localized granules or in a diffused form. They, however, did not differ from their parental strains in size and shape of yeast cells, fermentation and assimilation of sugars. Initially, the pink mutants were more resistant to amphotericin B, but upon passage on agar media for a prolonged period, they became as sensitive as the wild types. The pink strain produced less mycelium in vitro and in vivo, and were less virulent for mice, but more resistant to ultraviolet irradiation than the wild types.

The rôle of the host plant in the production of winged forms by two strains of the pea aphid, Acyrthosiphon pisum

Aphids of both strains of Acyrthosiphon pisum can produce alate daughters after experiencing a controlled period of contact with other aphids. This response is maternal, embryos being irrevocably determined as apterae or alatae very shortly before birth. Females of the green strain crowded soon after the onset of parturition did not produce many alate offspring when reared exclusively on seedlings of the bean Vicia faba. When the insects fed on mature bean leaves after the 24 hr crowding treatment the proportion of alatae in their progenies was more than doubled. Besides being influenced by the diet imbibed after the crowding experience, the maternal response was conditioned by the host plant offered the test insects during their larval development. Bean seedling host plants decreased the proportion of alatae subsequently produced, while mature bean leaves had the reverse effect. The production of numerous alatae in response to the combined influence of crowding and a mature leaf host was rapidly halted when the parent aphids were transferred to bean seedlings. Often all larvae subsequently deposited became apterae. Aphids of the pink strain studied here were particularly sensitive to contact with other aphids, and readily produced large numbers of alatae even when feeding on bean seedlings. Nevertheless, more alatae appeared in the progenies of crowded females caged on mature bean leaves. Females of both strains reared singly on bean seedlings prior to the onset of parturition started to deposit alate daughters within 72 hr of being transferred without crowding to mature leaves. Progressively greater numbers of these forms were produced as larviposition continued. This response appeared earlier in the case of females reared singly on mature leaves from birth onwards. Two varieties of the pea Pisum sativum had a particularly pronounced effect on alata-production by females of the green strain. Regardless of whether the aphids imbibed sap from pea seedlings before or after a crowding treatment, the majority produced no alate progeny whatsoever. The response of the pink aphids was not as easily influenced by this factor. When previously crowded apterae of the green strain reproduced on mature pea leaves, a response developed of comparable intensity to that of insects confined on bean seedlings. Fewer alatae were produced by females caged on excised mature pea leaves fed with glucose and amino acids than on leaves fed with a pure glucose solution.

The role of crowding in the production of winged forms by two strains of the pea aphid, Acyrthosiphon pisum

Apterous females of both strains of Acyrthosiphon pisum responded to being crowded by including some alate daughters in their progenies. Females of the pink strain produced many more alate offspring in response to this stimulus than did the green forms. The proportion of alatae in the progenies of crowded females of both strains was dependent upon both the duration of the treatment and the density of the aphids when crowded. Whereas two individuals of the pink strain when confined together for 24 hr induced a strong response in each other, aphids of the green strain produced few alatae unless confined in groups of 10. Control aphids starved for 24 hr in isolation gave no response. However, winged forms occasionally appeared, apparently randomly, in the offspring of unstarved but isolated pink females. Similar females of the green strain produced only apterous daughters. Larvae of both strains are irrevocably determined as alatae or apterae before their birth. No post-natal response to tactile stimulation or any other environmental factor could be demonstrated. Determination probably takes place shortly prior to birth. The maternal response of the green forms can be triggered by crowding adult females. This treatment, when applied to aphids of this strain prior to the larval-adult ecdyses, does not induce the production of alatae. However, fourth instar, and even third instar, apterae of the pink strain respond to a crowding stimulus by ultimately producing numerous alate daughters. The response to crowding can be ascribed to tactile stimulation only. Removal of antennae diminished the response to crowding considerably. Slow-motion film sequences have shown individuals of the pink strain to be particularly sensitive to encounters with other aphids. A 24-hr period of contact with the rough walls of a confining muslin bag induced in isolated pink aphids a response as intense as that of aphids subjected to any crowding treatment. Females of the green strain did not respond to this stimulus, and cine films show them to be much less sensitive to contact with other aphids than the pink forms.