http://digitalrepository.fccollege.edu.pk/handle/123456789/6 School of Life Sciences 2026-06-30T02:35:07Z http://digitalrepository.fccollege.edu.pk/handle/123456789/2784 Title: Assessment of Anticancer and Antimicrobial Potential of Bioactive Metabolites and Optimization of Culture Conditions of Pseudomonas aurantiaca PB-St2 for High Yields Authors: Mehnaz, Dr. Samina Abstract: The following study aimed to characterize the biological potential of the purified compounds of Pseudomonas aurantiaca PB-St2. Optimization of temperature and incubation time of 32o C and 72 h yielded the highest crude extract weight and optical density of bacterial culture. HPLC analysis of the crude metabolite extract (purified using gravitational column chromatography) showed three fractions named as PC1, PC2, and PC3. HPLC-purified fractions were subjected to LC-MS/MS analysis and the data was compared using reference library. Fraction PC1 was identified as mupirocin, PC2 as phenazine-1-carboxylic acid (PCA), and PC3 as the mixture of three compounds including pyoluteorin, PCA and 2-hydroxyphenazine (2-OH-phz). Fungicidal potential of the purified compounds was assessed against phytopathogens including Fusarium equiseti, Fusarium incarnatum, Alternaria alternata, and Colletotrichum falcatum. Fraction PC3 showed the highest fungicidal activity of ~89%, whereas, the least antifungal activity (~27%) was noted for mupirocin. Antibacterial activity of the purified compounds against Gram-positive pathogen Bacillus cereus, and Gram-negative pathogens Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella oxytoca was also assessed. Fraction PC3 demonstrated the highest antibacterial activity against B. cereus and P. aeruginosa showing 1.8 cm, and 0.9 cm zones of inhibition, respectively. Against K. oxytoca and S. enterica, the antibacterial activity of PB-St2 crude extract was slightly higher than the fraction PC3. The fraction PC3 also demonstrated the highest IC50 against HepG-2 and SF767 cancer cell lines at 25 μg and 20 μg concentrations, respectively. The multifaceted attributes of P. aurantiaca PB-St2 make it an ideal candidate for agricultural and pharmacological applications. Description: N/A 2024-03-31T00:00:00Z http://digitalrepository.fccollege.edu.pk/handle/123456789/2783 Title: Pyrrolnitrin is integral for antimicrobial activity and phenazine biosynthesis of Pseudomonas chlororaphis strains Authors: Mehnaz, Dr. Samina Abstract: Pseudomonas species are recognized for producing a diverse array of microbial metabolites with significant potential across various fields. Pyrrolnitrin (PRN), a halogenated metabolite based on phenylpyrrole, exhibits potent antibiotic properties. This research aimed to examine the influence of pyrrolnitrin on the antagonistic properties of Pseudomonas chlororaphis strains PB-St2, FS2, and RP4. Mutants of P. chlororaphis were generated by inhibiting prnA using two distinct suicide vectors, pEX18Tc and pKC1132. Analysis via high performance liquid chromatography (HPLC) revealed that pyrrolnitrin production was completely eliminated in the pKC1132 mutant and decreased by 82.5% in the pEX18Tc mutant. Both mutants also exhibited reduced phenazine production, with pKC1132 mutants showing a 61.1% reduction in phenazine-1-carboxylic acid (PCA) and pEX18Tc-induced mutants displaying a 39.9% decrease in PCA. To further elucidate the dependence of pyrrolnitrin production on other regulatory elements, the complete prnABCD operon with its native promoter was cloned and expressed in Escherichia coli (BL21). The antimicrobial potential of purified pyrrolnitrin was evaluated against fungal plant pathogens, human bacterial pathogens, and cancer cell lines (HepG-2 and SF767). The most pronounced inhibitory effect on Alternaria alternata was observed with 100 μg of pyrrolnitrin, resulting in an 82% reduction in spore formation followed by its effect on Aspergillus niger, causing a 75% decrease in spore production. Pyr- rolnitrin’s antibacterial activity produced inhibition zones of 1.8 cm against Salmonella enterica, 3.4 cm against Bacillus cereus, and 1.4 cm against Staphylococcus sp. at a concentration of 75 μg. The antiproliferative effects of pyrrolnitrin on cancer cell lines demonstrated 50% inhibition of both HepG-2 and SF767 cell lines at concentrations of 15 μg and 25 μg, respectively. Pyrrolnitrin exhibited significant antifungal and antibacterial activities, as well as cytotoxic effects on cancer cell lines, indicating its potential as both a biocontrol agent and therapeutic compound. Description: N/A 2025-05-14T00:00:00Z http://digitalrepository.fccollege.edu.pk/handle/123456789/2782 Title: Biocontrol potential of Bacillus sp. against indigenous fungal phytopathogens of potato and sugarcane Authors: Mehnaz, Dr. Samina Abstract: Fungal diseases pose significant threats to crops, necessitating effective control measures to mitigate their impact on agricul- tural productivity. In this study, fungal pathogens affecting sugarcane and potato crops in Pakistan were investigated. Morphological and molecular characterization revealed the presence of Fusarium sacchari and Ceratocystis sp. in sugar- cane samples, and Epicoccum nigrum and Alternaria alternata in potato samples. Effective biocontrol of these pathogens was shown by two corn rhizobacteria, i.e. Bacillus subtilis (MUN-1) and Bacillus velezensis (MUN-15). Both strains exhib- ited significant inhibition of fungal growth in vitro with Bacillus subtilis demonstrating higher efficacy. Gene-specific PCR anal- ysis confirmed the presence of surfactin and iturin genes responsible for antifungal metabolite production in these bac- teria in addition to VOCs. To the best of our knowledge, this is the first report of Epicoccum nigrum infecting potato plants in Pakistan. Implementing such eco-friendly strategies can contribute to the reduction of chemical inputs in agriculture ensuring crop health and productivity. Description: N/A 2025-02-02T00:00:00Z http://digitalrepository.fccollege.edu.pk/handle/123456789/2781 Title: Molecular and functional analysis of a putative pyocin S9, with endonuclease activity from P. chlororaphis subsp aurantiaca PB-St2 Authors: Mehnaz, Dr. Samina Abstract: Pyocins are bacteriocins which are explicitly associated with pseudomonads. In this study, the genome mining and in- depth sequence analysis identified three similar S9-like (a, b, and c), an S3-like (d) and one R-type pyocin systems from P. chlororaphis subsp aurantiaca PB-St2. The phenotypic screening of bacteriocin production by PB-St2 indicated narrow- spectrum bactericidal activity against closely related Pseudomonas species i.e., Pseudomonas aeruginosa PAi, PAc1, PAc3, PAc4; Pseudomonas fluorescens Psi-RS1 and Pseudomonas kilonensis OSRS3. Herein, the proposed pyocin S9c was further selected for molecular and functional characterization. The presumptive N-terminal receptor binding domain of candidate system lacks significant similarity with any characterized HNH-type pyocin S DNases from P. aeruginosa. In contrast, the cytotoxic domain showed 53% sequence similarity with pyocin S8 and 70% to pyocin S9. Thus, pyocin S9c was suggested as an isoform under the Class I DNase (H-N-H) family in pyocin S9 cluster, commonly found in P. chlororaphis subsp. aurantiaca and P. chlororaphis subsp aureofaciens strains. Molecular screening of the pyocin S9c system revealed its presence in 6 out of 7 tested strains of P. chlororaphis subsp. aurantiaca GS1, GS3, GS4, GS6, ARS38, FS2 and one P. chlororaphis RP4 relative strains, isolated from diverse plant hosts. The 1.59 kb fragment consisting of two structural genes of pyocin-immunity operon (S9c) in P. aurantiaca PB-St2 were cloned in pET28a(+) and expressed in Escherichia coli BL21 DE3 (pLysS) strain as a fusion protein with histidine tag. The recombinant cytotoxic protein of pyocin S9c operon was purified with N-term His-tag with a molecular weight of ≈50 kDa. The identity of target protein was affirmed by tandem mass spectrometry analysis. The purified cytotoxic protein was active against P. chlororaphis subsp. aurantiaca GS7, with a minimum inhibitory concentration of 12.5 μg/ml. The mechanism of cytotoxicity was affirmed as a metal-dependent endonuclease by evidence of non-specific hydrolysis of pTZ57R plasmid isoforms. These results indicate that pyocin S9c can contribute to the rhizo-competence of this strain in plant-associated natural habitats, occupied by related Pseudomonas strains. 2025-04-26T00:00:00Z