Hillol Chakdar

Bacterial xylanases: biology to biotechnology

In this review, a comprehensive discussion exclusively on bacterial xylanases; their gene organization; different factors and conditions affecting enzyme yield and activity; and their commercial application have been deliberated in the light of recent research findings and extensive information mining. Improved understanding of biological properties and genetics of bacterial xylanase will enable exploitation of these enzymes for many more ingenious biotechnological and industrial applications.

Deciphering Diversity of Salt-Tolerant Bacilli from Saline Soils of Eastern Indo-gangetic Plains of India

The intensive use of chemical fertilizers, monoculture and irrigation with surface saline water has resulted in the deterioration of soil health by enhancing the level of salinity in the Eastern Indo-Gangetic Plains of India. Therefore, diversity of halotolerant bacteria adapted to that environment and possessed the ability to produce plant growth hormones was explored, that could be used for salt stress amelioration. The 16S rRNA gene sequencing and fatty acid methyl ester (FAME) were used for diversity analysis of salt-tolerant bacilli. Among the 95 isolates, 55 strains showed plant growth promotion traits, production of industrially important enzymes (amylase, protease and cellulase) and tolerance to more than 4% NaCl. Using partial 16S rRNA sequences and FAME comparisons, 21 different species of Bacillus and Bacillus-derived genera were identified, viz. Bacillus megaterium, B. subtilis, B. licheniformis, B. firmus, B. horikoshii, B. pumilus, Bacillus sp., B. safensis, B. thuringiensis, B. simplex, B. agri, B. flexus, B. oceanisediminis, B. cereus, B. arsenicus, Paenibacillus dendritiformis, Lysinibacillus sp., L. sphaericus, B. marisflavi, Terribacillus sp., and B. mycoides. These isolates possess the ability to tolerate high salt, form endospores, withstand harsh environments, and also have the potential for plant growth promotion, which could be useful in formulation of new inoculants to enhance the availability of nutrients for crop growth under saline conditions.

Isolation and characterization of biosurfactant producing Bacillus sp. from diesel fuel-contaminated site

Among hydrocarbon pollutants, diesel oil is a complex mixture of alkanes and aromatic compounds which are often encountered as soil contaminants leaking from storage tanks and pipelines or as result of accidental spillage. One of the best ecofriendly approaches is to restore contaminated soil by using microorganisms able to degrade those toxic compounds in a bioremediation process. In the present study, nineteen bacteria were isolated by enrichment culture technique from diesel spilled soil collected from electric generator shed of NBAIM, Mau. All the isolates were subjected to screening for lipase production and twelve isolates were found to be positive for lipase. When the isolates were screened for biosurfactant production using CTAB-methylene blue agar plates, only one isolate viz. 2NBDSH3 was found positive which was found to be phylogenetically closely related with Bacillus flexus. Despite having low emulsification index, the bacterium could degrade 88.6% of diesel oil in soil. Biosurfactant from the isolate was extracted and characterized through infra-red spectroscopy which indicated its possible lipopeptide nature which was further supported by strong absorption in UV range in the UV-Vis spectrum. The results of the present study indicated that the isolate either does not produce any bioemulsifier or produces very low amount of emulsifier rather it produces a lipopeptide biosurfactant which helps in degradation of diesel oil by lowering the surface tension. The bacterium thus isolated and characterized can serve as a promising solution for ecofriendly remediation of bacterium diesel contaminated soils.

Nanotechnology for the Detection and Diagnosis of Plant Pathogens

Rapid detection technologies with high sensitivity and selectivity for plant pathogens are essential to prevent disease spread with minimal loss to crop production and food quality assurance. Traditional laboratory techniques such as microscopic and cultural techniques are time-consuming and require complex sample handling. Immunological and molecular techniques are advanced but have some issues related to rapidity and signal strength. In this context, integration of immunological and molecular diagnostics with nanotechnology systems offers an alternative where all detection steps are done by a portable miniaturized device for rapid and accurate identification of plant pathogens. Further, nanomaterial synthesis by utilizing functionalized metal nanoparticles as a sensing component offer several desirable features required for pathogen detection. The sensitive nature of functionalized nanoparticles can be utilized to design phytopathogen detection devices with smart sensing capabilities for field use. This chapter provides an overview of the application of nanotechnology in the field of microbial diagnostics with special focus on plant pathogens.

Comparison of molecular and phenetic typing methods to assess diversity of selected members of the genus Bacillus

A comparison of different molecular typing methods viz. ERIC-PCR, BOX-PCR and ARDRA along with carbohydrate utilization pattern was carried out to analyze their discriminatory power and suitability for assessing diversity of selected Bacillus isolates. ERIC-PCR generated 61 bands ranging from 0.56 to 3.9 kb while BOX-PCR resulted in 127 bands ranging from 0.16 to 3.9 kb. Restriction analysis of 16S rDNA with AluI and HaeIII produced 56 and 67 bands ranging from 0.14 to 0.54 and 0.12 to 0.96, respectively. Clustering of isolates based on the ERIC, BOX and ARDRA pattern clearly showed the superiority of the former two methods to reveal the intrageneric and intraspecific diversity. Carbohydrate utilization pattern showed that most preferred sugar was Fructose while Xylose, Rhamnose and D-Arabinose were least preferred by the isolates used for the study. Clustering based on carbohydrate utilization was also able to differentiate among the isolates which showed 100% similarity based on ARDRA profiles. This study clearly shows that typing methods exploiting the repetitive elements distributed over the genome are more useful for assessing genetic diversity. Moreover, metabolic diversity of the bacterial groups may also be useful instead of using single locus specific marker systems for revealing the diversity.

Deciphering the salinity adaptation mechanism in Penicilliopsis clavariiformis AP, a rare salt tolerant fungus from mangrove.

Penicilliopsis clavariiformis AP, a rare salt tolerant fungus reported for the first time from India was identified through polyphasic taxonomy. Scanning electron microscopy showed that the fungus has unique features such as biverticillate penicilli bearing masses of oval to ellipsoidal conidia. The fungus has been characterized for salt tolerance and to understand the relevance of central carbon metabolism in salt stress adaptation. It showed optimal growth at 24 °C and able to tolerate up to 10% (w/v) NaCl. To understand the mechanism of adaptation to high salinity, activities of the key enzymes regulating glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle were investigated under normal (0% NaCl) and saline stress environment (10% NaCl). The results revealed a re‐routing of carbon metabolism away from glycolysis to the pentose phosphate pathway (PPP), served as a cellular stress‐resistance mechanism in fungi under saline environment. The detection and significant expression of fungus genes (Hsp98, Hsp60, HTB, and RHO) under saline stress suggest that these halotolerance conferring genes from the fungus could have a role in fungus protection and adaptation under saline environment. Overall, the present findings indicate that the rearrangement of the metabolic fluxes distribution and stress related genes play an important role in cell survival and adaptation under saline environment.

DNA Barcoding for Diagnosis and Monitoring of Fungal Plant Pathogens

Plant pathogenic fungi cause significant economic crop yield losses every year. Proper identification up to species level is a critical initial step in any investigation of plant infection, whether it is research driven or compelled by the need for rapid and accurate diagnostics during disease outbreak. Further, it is also helpful in making decision with respect to the monetary loss and investment to follow necessary disease management practices. The recent developments of DNA barcoding technology have drastically translated the epitome of species identification and show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, ecological studies, diagnostics and monitoring of fungal plant pathogens. This chapter provides a vision on the current use and impact of DNA barcoding approaches in diagnosis and monitoring of fungal plant pathogens. Moreover, an effort has been put forward to understand various marker genes associated with barcode process, their suitability, limitation and applicability in diagnostic and monitoring of fungal plant pathogens.

Cyanobacterial Phycobilins: Production, Purification, and Regulation

The cyanobacteria are a diverse group of prokaryotic organisms that carry out oxygenic photosynthesis and are thought to be responsible for the oxygenation of our atmosphere. Like red algae and cryptomonads, cyanobacteria also contain phycobiliproteins (PBPs) which serve as major accessory pigments during photosynthesis. PBPs are large water-soluble supramolecular protein aggregates involved in light harvesting and can be divided broadly into three classes, viz., phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC) based on their spectral properties. PBPs have been extracted and purified from Spirulina spp., Synechococcus sp., Oscillatoria sp., etc., and produced commercially from Spirulina platensis, Anabaena sp., and Galdieria sulphuraria. Since cyanobacteria exhibit wide variations in nutrient availability, light intensity, light quality (wavelength), temperature, water activity, etc., these variations also result in altered metabolic activity of these organisms as a result of differential expression of different genes. The expression of phycobiliprotein coding genes is also accordingly modulated to adapt to a particular condition. Many workers have reported changes in phycobilisome structure and expression of cpc genes in response to light quality, light quantity, and nutrients like nitrogen, sulfur, etc. The composition and function of phycobiliproteins in cyanobacteria have also been reported to change under stress conditions. In the present paper, we have reviewed the production, purification, and regulation of cyanobacterial phycobilins including their importance in the commercial sector, as they have several applications as natural dyes in food and cosmetic industry, immunological assays, health-promoting properties, and broad range of other pharmaceutical applications.

Halotolerant Exiguobacterium profundum PHM11 Tolerate Salinity by Accumulating L-Proline and Fine-Tuning Gene Expression Profiles of Related Metabolic Pathways

Salinity stress is one of the serious factors, limiting production of major agricultural crops; especially, in sodic soils. A number of approaches are being applied to mitigate the salt-induced adverse effects in agricultural crops through implying different halotolerant microbes. In this aspect, a halotolerant, Exiguobacterium profundum PHM11 was evaluated under eight different salinity regimes; 100, 250, 500, 1000, 1500, 2000, 2500, and 3000 mM to know its inherent salt tolerance limits and salt-induced consequences affecting its natural metabolism. Based on the stoichiometric growth kinetics; 100 and 1500 mM concentrations were selected as optimal and minimal performance limits for PHM11. To know, how salt stress affects the expression profiles of regulatory genes of its key metabolic pathways, and total production of important metabolites; biomass, carotenoids, beta-carotene production, IAA and proline contents, and expression profiles of key genes affecting the protein folding, structural adaptations, transportation across the cell membrane, stress tolerance, carotenoids, IAA and mannitol production in PHM11 were studied under 100 and 1500 mM salinity. E. profundum PHM11 showed maximum and minimum growth, biomass and metabolite production at 100 and 1500 mM salinity respectively. Salt-induced fine-tuning of expression profiles of key genes of stress pathways was determined in halotolerant bacterium PHM11.

Genome-Wide Analysis of Microsatellites in Alternaria arborescens and Elucidation of the Function of Polyketide Synthase (PksJ)

Microsatellites or simple sequence repeats (SSRs) have been the most widely applied class of molecular markers used in genetic studies, having applications in genetic conservation, population studies, as well as diagnostics of fungi. Mining and analysis of SSRs of the whole genome sequence have been carried out in this study for the fungus Alternaria arborescens causing early blight of tomato and well known for producing mycotoxins like alternariol (AOH), alternariol monomethyl ether (AME), etc. A total of 4097 microsatellites were identified in A. Arborescens genome. Contig 1 was identified as the most SSR-rich region which was further analyzed to correlate the presence of SSRs with different biological processes. A total of 246 putative genes were predicted in this study and KEGG pathway analysis of 155 predicted genes indicated that SSRs can be linked with important metabolic pathways, molecular functioning, signal transduction, and cellular processes. The prediction of fungal mycotoxin inducer gene Polyketide synthase (PksJ) linked with SSR in this study may be a potential candidate participating in oncogenic signal transduction in human. Our study is the first report of PksJ gene in A. arborescens, a precursor of AOH and AME.