Amer Jamil

Molecular cloning of fungal xylanases: an overview

Xylanases have received great attention in the development of environment-friendly technologies in the paper and pulp industry. Their use could greatly improve the overall lignocellulosic materials for the generation of liquid fuels and chemicals. Fungi are widely used as xylanase producers and are generally considered as more potent producers of xylanases than bacteria and yeasts. Large-scale production of xylanases is facilitated with the advent of genetic engineering. Recent breakthroughs in genomics have helped to overcome the problems such as limited enzyme availability, substrate scope, and operational stability. Genes encoding xylanases have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Owing to the industrial importance of xylanases, a significant number of studies are reported on cloning and expression of the enzymes during the last few years. We, therefore, have reviewed recent knowledge regarding cloning of fungal xylanase genes into various hosts for heterologous production. This will bring an insight into the current status of cloning and expression of the fungal xylanases for industrial applications.

Gene Expression Profiling of Plants under Salt Stress

Soil salinity is among the leading environmental stresses affecting global agriculture, causing billions of dollars in crop damages every year. Regardless of the cause, ion toxicity, water deficit, or nutritional imbalance, high salinity in the root zone severely impedes normal plant growth and development, resulting in reduced crop productivity or crop failure. Development of salt-tolerant cultivars is an attractive and economical approach to solving this problem. Although several salt-tolerant plant genotypes have been developed through transgenic approaches, often they have failed or exhibited limited success under field saline conditions. This is due to several reasons, including the fact that plant growth and development under saline conditions in the field is often influenced by cumulative effects of multiple environmental stresses and genetic factors, which may not have been considered during the development of salt-tolerant transgenic plants. Adoption of inappropriate screening techniques or selection criteria may also lead to selection of genotypes that may not be stress tolerant in a real sense. In most plant species, salt tolerance is a genetically complex trait, often modulated by multiple biosynthetic and signaling pathways. Cross-talks among various stress-controlling pathways have been observed under salt stress, many of which are regulated by transcription factors. Thus, a comprehensive knowledge of the up- and downregulating genes under salt-stress is necessary, which would provide a better understanding of the interactions among pathways in response to salt stress. Attaining such knowledge is a good step toward successful development of salt-tolerant crop cultivars. To this end, DNA microarray technology has been employed to study expression profiles in different plant species and at varying developmental stages in response to salt stress. As a result, large-scale gene expression profiles under salt stress are now available for many plant species, including Arabidopsis, rice, barley, and ice plant. Examinations of such gene expression profiles will help understand the complex regulatory pathways affecting plant salt tolerance and potentially functional characterization of unknown genes, which may be good candidates for developing plants with field salt tolerance. In this article, we review and discuss the current knowledge of plant salt tolerance and the extent to which expression profiling has helped, or will help, a better understanding of the genetic basis of plant salt tolerance. We also discuss possible approaches to improving plant salt tolerance using various tools of biotechnology.

Effect of biofilm formation on the excretion of Salmonella enterica serovar Typhi in feces

OBJECTIVES We hypothesized that Salmonella enterica serovar Typhi (S. Typhi) with higher biofilm and capsule production capability are more able to survive continuously in typhoid patients/carriers, with subsequent prolonged shedding in feces. METHODS Bacterial cell release from biofilm (produced in vitro and confirmed by specific staining and electron microscopy) and comparative cytotoxicity were studied on Caco2 cells. Functionality of the biofilm diffusion barrier was tested against ciprofloxacin. Biofilm production was graded and semi-quantified as -, +, ++, +++, and ++++. RESULTS Out of 30 isolates, 23 produced biofilm. The average post-treatment detection of S. Typhi in blood was 7-13 days and in stool was 13-32 days. A fall in cell count from 10⁴ to approximately 10¹ over the course of 3 days as compared to total elimination of planktonic cells in 16 h after ciprofloxacin application substantiated the protective role of biofilm. Lactic dehydrogenase release ranged from 38% in non-biofilm producers to 97% in the highest biofilm producers, indicating increased pathogenic behavior. CONCLUSIONS The period of S. Typhi clearance from typhoid patients after recovery was found to be directly related to biofilm production capability.

Chemical changes during ensiling of sudax fodder with broiler litter

Abstract Sudax fodder was ensiled in large trench silos with or without the addition of 30% broiler litter and 6% molasses. The samples were analysed for pH, lactic acid, nitrogen and fiber fraction at 0- and 5-day interval till 40 days. The silage containing broiler litter had higher initial and subsequent pH due to high ammonia concentration. pH of both silages had the largest drop during the first 5 days of ensiling, with corresponding increase in lactic acid. pH continued decreasing until 10 days in silage without litter and 15 days in silage containing litter. Lactic acid continued increasing in silage without litter until 15 days and with litter until 25 days of ensiling. The addition of broiler litter resulted in significantly decreased content of NDF, hemicellulose and cellulose and increased content of ADF. Ensiling period had significant effect on all the fiber fractions. Total-N content increased slightly during the ensiling process but protein-N decreased considerably at the fifth day of ensiling with simultaneous increase in ammonia-N. Later on, there was a slight reversal but it never corresponded with the initial values.

Molecular cloning and comparative sequence analysis of fungal β-Xylosidases

Commercial scale degradation of hemicelluloses into easily accessible sugar residues is practically crucial in industrial as well as biochemical processes. Xylanolytic enzymes have a great number of possible applications in many biotechnological processes and therefore, these enzymes are continuously attracting the attention of scientists. Due to this fact, different β-Xylosidases have been isolated, purified and characterized from several bacteria and fungi. Microorganisms in this respect have gained much momentum for production of these significant biocatalysts with remarkable features. It is difficult to propagate microorganisms for efficient and cost-competitive production of β-Xylosidase from hemicelluloses due to expensive conditions of fermentation. The screening of new organisms with an enhanced production of β-Xylosidases has been made possible with the help of recombinant DNA technology. β-Xylosidase genes haven been cloned and expressed on large scale in both homologous and heterologous hosts with the advent of genetic engineering. Therefore, we have reviewed the literature regarding cloning of β-Xylosidase genes into various hosts for their heterologous production along with sequence similarities among different β-Xylosidases. The study provides insight into the current status of cloning, expression and sequence analysis of β-Xylosidases for industrial applications.

Effect of ensiling sudax fodder with broiler litter and Candida yeast on the changes in different fiber fractions

Sudax fodder was ensiled in laboratory silos without or with the addition of 20, 30 or 40% broiler litter and 6% molasses, each without or with yeast inoculant (Candida utilus). Different fiber fractions were analyzed at the start of the ensiling and at 10-day intervals thereafter for 40 days. NDF, ADF, cellulose and lignin increased and cell contents decreased as a result of fermentation during ensiling. The addition of increasing levels of broiler litter not only decreased NDF, hemicellulose and cellulose, with a simultaneous increase in ADF content, but also extended the time course of fermentation during ensiling. The yeast inoculant failed to produce any significant effect on the fermentation characteristics or on any of the fiber fractions of the silage.

Activity‐guided isolation of a novel protein from Croton tiglium with antifungal and antibacterial activities

This study describes the activity‐guided isolation and purification of a novel antimicrobial protein from the seed of Croton tiglium Linn. Purification was carried out by (NH4)2SO4 precipitation, gel filtration and DEAE‐cellulose ion‐exchange chromatography. Antifungal and antibacterial activities were determined after each purification step. SDS‐polyacrylamide gel electrophoresis revealed that the purified protein was a monomer with molecular mass of 50 kDa. This is a first report on purification of a protein from Croton tiglium, which possesses a strong and broad spectrum antimicrobial activity. Copyright

De novo sequencing and transcriptome analysis of female venom glands of ectoparasitoid Bracon hebetor (Say.) (Hymenoptera: Braconidae)

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom from the ectoparasitoid Bracon hebetor, a tiny wasp that attacks larval pest insects of field and stored products and is thus a potential insect control agent, has not described the full complement and composition of these biomolecules. To have a comprehensive picture of genes expressed in the venom glands of B. hebetor, a venom gland transcriptome was assembled by using next generation sequencing technologies followed by de novo assemblies of the 10.81 M sequence reads yielded 22,425 contigs, of which 10,581 had significant BLASTx hits to know genes. The majority of hits were to Diachasma alloeum, an ectoparasitoid from same taxonomic family, as well as other wasps. Gene ontology grouped the sequences into molecular functions in which catalytic activity with 42.2% was maximum, cellular components in which cells with 33.8% and biological processes among which metabolic process with 30% had the most representatives. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Full length ORFs of Calreticulin, Venom Acid Phosphatase Acph-1 like protein and arginine kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. Our report is the first to characterize components of the B. hebetor venom glands that may be useful for developing control tools for insect pests and other applications.

Comparative sequence analysis of citrate synthase and 18S ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi

A mutation was induced in Aspergillus niger wild strain using ethidium bromide resulting in enhanced expression of citric acid by three folds and 112.42 mg/mL citric acid was produced under optimum conditions with 121.84 mg/mL of sugar utilization. Dendograms of 18S rDNA and citrate synthase from different fungi including sample strains were made to assess homology among different fungi and to study the correlation of citrate synthase gene with evolution of fungi. Subsequent comparative sequence analysis revealed strangeness between the citrate synthase and 18S rDNA phylogenetic trees. Furthermore, the citrate synthase movement suggests that the use of traditional marker molecule of 18S rDNA gives misleading information about the evolution of citrate synthase in different fungi as it has shown that citrate synthase gene transferred independently among different fungi having no evolutionary relationships. Random amplified polymorphic DNA (RAPD-PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product. Abbreviations CS - Citrate synthase, CA - Citric acid, RAPD - Random amplified polymorphic DNA, TAF - Total amplified fragments, PAF - Polymorphic amplified fragments, CAF - Common amplified fragments.

Cloning of an intronlesscre1 gene fromChaetomium thermophilum

The expression of xylanases and cellulases is under carbon catabolite repression, a regulatory repression mechanism of transcription of these enzymes caused bycre1 gene. In the present study we isolatedcre1 partial gene from a thermophilic fungusChaetomium thermophilum ATCC 28076. The fungus was grown on Eggins and Pugh medium with glucose as a carbon source. Genomic DNA was isolated by two different methods and the integral DNA was subjected to polymerase chain reaction (PCR) for the amplification ofcre1 partial gene sequence. The PCR product was ligated into pTZ57R/T vector and transformed in E.coli TOP 10 strain. cDNA from total RNA was utilized as template for RT-PCR analysis, which confirmed the presence of an intronless cre1 gene in the model organism. This is the first report on isolation ofcre1 gene fragment in C.thermophilum.