Nafees Bacha

Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A

Ochratoxin A (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthase gene aoks1, cloned by using gene walking approach. The predicted amino acid sequence of the 2kb clone display 34-60% similarities to different polyketide synthase genes including lovastatine biosynthesis gene lovb in A. terreus, compactin biosynthesis gene mlcA in Penicillium citrinum and OTA biosynthesis gene otapksPN in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1 gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174.

Regulation and role of a STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum

In phytopathogenic fungi, STE12‐like genes encode transcription factors essential for appressorium‐mediated host penetration. However, their regulation and downstream targets are still unknown. In the present study, a STE12‐like gene (CLSTE12) from Colletotrichum lindemuthianum was isolated. We identified a spliced variant whose expression was negatively regulated during early stages of pathogenesis, whereas the correctly spliced mRNA remained expressed up to the penetration step, suggesting distinct roles for these two transcripts. Indeed, the full‐length sequence was able to complement a yeast STE12 mutant, whereas overexpression of the transcript variant had a dominant‐negative effect on yeast invasive growth and C.u2003lindemuthianum pathogenicity. To further investigate the downstream genes that could be regulated by CLSTE12, disruption mutants were generated. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to polystyrene. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p, which was absent from the mutants. Clsp1p belongs to a new family of wall‐associated proteins only found in euascomycetous fungi. Overall, these results suggest that the activity of CLSTE12 can be modulated by a regulated alternative splicing mechanism and that this factor is involved in the production of cell surface proteins and host cell wall degrading enzymes.

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298bp polyketide synthase gene aomsas has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40-56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant aoDeltamsas of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDeltamsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone.

Fungi as chemical industries and genetic engineering for the production of biologically active secondary metabolites

Fungi is somewhere in between the micro and macro organisms which is a good source of producing biologically active secondary metabolites. Fungi have been used as tool for producing different types of secondary metabolites by providing different nutrients at different laboratory conditions. The fungi have been engineered for the desired secondary metabolites by using different laboratory techniques, for example, homologous and heterologous expressions. This review reported how the fungi are used as chemical industry for the production of secondary metabolites and how they are engineered in laboratory for the production of desirable metabolites; also the biosynthetic pathways of the bio-organic-molecules were reported.

Polyketide synthase gene aolc35-12 controls the differential expression of ochratoxin A gene aoks1 in Aspergillus westerdijkiae

Ochratoxine A (OTA), a potential human carcinogen is produced by several species of Aspergillus and Penicillium, including Aspergillus westerdijkiae. In this study a putative polyketide synthase gene aolc35-12 has been partially cloned from A. westerdijkiae. The predicted amino acid sequence of the 3.22 kb clone was found to have a high degree of similarity to other previously identified polyketide synthase genes from various OTA-producing fungi including Aspergillus ochraceus, Aspergillus niger, Aspergillus carbonarius and Penicillium nordicum. The aolc35-12 gene was disrupted and inactivated by insertion of Escherichia coli hygromycin B phosphotransferase gene, which resulted in an OTA negative mutant aoΔlc35-12. Genetic complementation confirmed aolc35-12 as OTA-polyketide synthase gene. Furthermore, study of the differential expression of aolc35-12 and a previously identified OTA-polyketide synthase gene, i.e. aoks1, in the wild-type A. westerdijkiae and aoΔlc35-12 mutant revealed that aolc35-12 could...

Characterization of a cytochrome P450 monooxygenase gene involved in the biosynthesis of geosmin in Penicillium expansum

Geosmin is a terpenoid, an earthy-smelling substance associated with off-flavors in water and wine. The biosynthesis of geosmin is well characterized in bacteria, but little is known about its production in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. Herein, we describe the characterization of “gpe1”, a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in this species. A gpe1knockout mutant of P. expansum M2230 lost the capacity to produce geosmin, while the genetically complemented mutant restored it. The deduced gpe1 protein sequence shows identities with other cytochrome P450 monooxygenases involved in diterpene biosynthesis. These enzymes catalyze the addition of hydroxyl groups to the diterpene compounds. gpe1protein could work in the same way, with sesquiterpenes as substrates. This gene seems to be only present in geosmin-producing Penicillium species. To our knowledge, this is the first characterization of a fungal gene encoding an enzyme involved in geosmin biosynthesis.

Study of the genetic traits associated with antibiotic resistance in Staphylococcus aureus isolated from skin wards of Khyber Pakhtunkhwa, Pakistan

Abstract Objective To investigate the prevalence of Staphylococcus aureus ( S. aureus ) isolated from skin wards of the hospitals of Khyber Pakhtunkhwa, its resistance against various commonly and commercially available antibiotics, as well as different genetic traits of resistance and their correlations with the phenotypic visible resistance. Methods In the present study a simple PCR technique were used to investigate the genetic traits of resistance in S. aureus isolated from skin wards of two major hospitals of Khyber Pakhtunkhwa, Pakistan. A total of 100 samples were collected from both the male and female, of which 50 were from patients site of infection and 50 from ward environment. Results These results demonstrated that the total prevalence of S. aureus both in ward as well as in patients was 48%. The S. aureus prevalence was the highest in female patients (50%) followed by ward environment (29%) and then male patients (21%). The antibiotic sensitivity tests revealed that the highest (91.6% isolates) sensitivity was shown to imipenem. However, the highest resistance was found to be against penicillin (100% isolates) followed by cefotaxime (75% isolates). In addition, only 29% of the isolates were found to be resistant to methicillin. PCR technique based on the previously designed primers targeting different genetic traits of resistance revealed that 13 out of the 14 isolates resistant to methicillin were positive for mecA gene. blaZ Genetic traits were found in all isolates resistant to penicillin. The multi-drug resistance traits, vgaA and vgaB each was detected only in 12.5% of S. aureus isolates. The phenotypic character of antibiotic resistance is highly correlated to different genetic traits of resistance. Conclusions Based on our findings, it is concluded that antibiotic resistance in S. aureus strains is increasing day by day due to self-medications and medication by non-registered medical practitioners. Therefore, for quick and fast detection, we propose next-generation sequencing be utilized to screen for antibiotic resistance.

Development of a novel quantitative PCR assay as a measurement for the presence of geosmin‐producing fungi

To provide an efficient technique for monitoring the off‐flavoured fungal compound geosmin.

Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7.

Purpose: Escherichia coli (E. coli) O157:H7 is gram-negative enteric pathogen producing different types of Shiga toxin. This bacterium is the most corporate cause of haemorrhagic colitis in human. Administration of antibiotics (particularly sulfa drugs) against this pathogen is a debatable topic as this may increase the risk of uremic syndrome; especially in children and aged people. Around the world, microbiologists are in search of alternative therapeutic methods specially probiotics against this pathogen. In the present study, we have focused on the investigation of alternate bio-therapeutics (probiotics) for the treatment of patients infected with E. coli O157:H7. This study is based on the identification of colicin-producing gram-negative bacteria (particularly enterobacteriaceae) which can competently exclude E. coli O157:H7 from the gut of the infected individual. Materials and Methods: Hundred samples from human, animal faeces and septic tank water were analysed for nonpathogenic gram-negative rods (GNRs). Results: Out of these samples, 175 isolates of GNRs were checked for their activity against E. coli O157:H7. Only 47 isolates inhibited the growth of E. coli O157:H7, among which majority were identified as E. coli. These E. coli strains were found to be the efficient producers of colicin. Some of the closely related species i. e., Citrobacter sp, Pantoea sp. and Kluyvera sp. also showed considerable colicinogenic activity. Moreover, colicinogenic species were found to be nonhaemolytic, tolerant to acidic environment (pH 3) and sensitive to commonly used antibiotics. Conclusion: Nonhaemolytic, acid tolerant and sensitive to antibiotics suggests the possible use of these circulating endothelial cells (CEC) as inexpensive and inoffensive therapeutic agent (probiotics) in E. coli O157:H7 infections.