Manish Singh Kaushik

Linking the Physicochemical Properties with the Abundance and Diversity of Rhizospheric Bacterial Population Inhabiting Paddy Soil Based on a Concerted Multivariate Analysis of PCR-DGGE and RISA

ABSTRACT To unravel the existence of dominant bacterial population in the paddy fields of Eastern Uttar Pradesh, India and their relation to the prevailing soil physicochemistry using multivariate statistical analyses, a cumulative culture-independent 16S rRNA based Polymerase chain reaction-Denaturing gradient gel electrophoresis (PCR-DGGE) and a 16S-23S ribosomal intergenic spacer analysis (RISA) have been performed. Detrended correspondence analysis (DCA) and principal component analysis (PCA) biplot analyses were used to assess the relation between soil bacterial population and its physicochemistry. DCA analysis exhibited a strong dependence of bacterial existence on the soil physicochemical variables, such as organic matter, total nitrogen, inorganic nutrients, temperatures, and moisture status. Soil dehydrogenase activity (DHA) was assessed to check the metabolic activity of all soil samples which showed a range of 0.012–0.050 nmol TPF g−1 min−1 with significant variation (p < 0.01). Out of 96 bands excised, 45 different phylotypes were obtained using both techniques which elucidated the abundance of Cyanobacteria over other soil bacterial population. Scytonema sp., Leptolyngbya sp. and different uncultured cyanobacterial species were the major genera found. Profiling data obtained through PCR-DGGE and RISA were used in alpha diversity and rarefaction curve analysis suggested site 6 (Chandauli) as the most diversity rich site. Thus extensive dataset of weighted and unweighted variables generated through DGGE and RISA coupled with metabolic functioning of soil and multivariate analyses provided an excellent opportunity to map the soil microbial structure in paddy fields and their regulation with existing soil environment.

Regulation of organophosphate metabolism in cyanobacteria. A review

Cyanobacteria sense the environmental phosphate level and respond accordingly with the help of a two component regulatory system SphS-SphR orthologous to PhoR-PhoB of E. coli, where SphS act as a sensor kinase and SphR as a response regulator. Under phosphate limiting condition SphS-SphR regulates the expression of many genes including genes which do not have the direct role in metabolism and transport of phosphate. Thus there is some crosstalk mechanism which connects this regulatory system to the other metabolic processes. Different types of enzymes and transporters are expressed by cyanobacteria under phosphate limitation to release and transport the phosphate from different organic compounds present in the environment. Genes encoding these enzymes and transporters contain Pho boxes in their promoter region where SphR binds and regulate their expression under phosphate limitation. The machinery and mechanism of regulation is not uniform in cyanobacteria as it varies in different groups according to their evolutionary adaptations. This review article is summarizing the reports on machinery and mechanism of organophosphate metabolism in cyanobacteria.

Role of manganese in protection against oxidative stress under iron starvation in cyanobacterium Anabaena 7120

The cyanobacterium Anabaena sp. PCC 7120 was grown in presence and absence of iron to decipher the role of manganese in protection against the oxidative stress under iron starvation and growth, manganese uptake kinetics, antioxidative enzymes, lipid peroxidation, electrolyte leakage, thiol content, total peroxide, proline and NADH content was investigated. Manganese supported the growth of cyanobacterium Anabaena 7120 under iron deprived conditions where maximum uptake rate of manganese was observed with lower Km and higher Vmax values. Antioxidative enzymes were also found to be elevated in iron‐starved conditions. Estimation of lipid peroxidation and electrolyte leakage depicted the role of manganese in stabilizing the integrity of the membrane which was considered as the prime target of oxygen free radicals in oxidative stress. The levels of total peroxide, thiol, proline and NADH content, which are the representative of oxidative stress response in Anabaena 7120, were also showed increasing trends in iron starvation. Hence, the results discerned, clearly suggested the role of manganese in protection against the oxidative stress in cyanobacterium Anabaena 7120 under iron starvation either due to its antioxidative properties or involvement as cofactor in a number of antioxidative enzymes.

Ferric Uptake Regulator (FUR) protein: properties and implications in cyanobacteria

The Ferric uptake regulator (Fur) protein is a global iron regulator found in most prokaryotes. Although the Fur protein is involved in a variety of metabolic pathways, it is specifically known for the regulation of several iron responsive genes. It binds to the highly conserved sequences located in the upstream promoter region known as iron boxes, using ferrous ion as a co-repressor. Apart from that, the Fur protein is also directly/indirectly involved in a variety of other crucial physiological pathways. Hence, understanding the mechanism of action and the mechanistic pathways of iron regulation by Fur is necessary and important. The basic understanding of the functioning and properties of Fur protein along with its role, interaction and regulation at various levels in cyanobacteria has been discussed in detail.

Molecular phylogeny of heterotrophic nitrifiers and aerobic denitrifiers and their potential role in ammonium removal

To investigate the physiology and taxonomic composition of the key players of nitrification and denitrification processes in paddy fields, culture dependent and independent studies have been carried out. A total of 28 bacterial strains have been screened in which six were capable of reducing nitrate and nitrite as well as having significant ammonium removal potential. 16S rRNA‐PCR‐DGGE‐based molecular typing of enriched batch culture was done with time duration to explore and identify dominant and stable soil denitrifiers. Notably, three isolates namely PDN3, PDN19, PDN14 were found to be efficiently involved in the removal of 70.32, 71.46, and 81.50% of NH4+ and showed closest similarity (>98%) with Bacillus cereus, Bacillus subtilis, and Pseudomonas aeruginosa strains, respectively. The bacterial strain PDN14 showed maximum growth with highest ammonium removal rate (2.78 gN/(m3·h) has also been characterized based on nosZ gene which showed similarity to uncultured γ‐ Proteobacteria, P. aeruginosa sp. B3. Median joining (MJ) network and rRNA secondary structure have been analyzed for their detailed taxonomic diversity and derived haplotype‐based co‐occurrence. Results demonstrated that such strains can serve as good candidate for in situ nitrogen transformation in paddy soils and improvingly characterized by physiological and detailed phylogenetic approaches.

Toxicity of biocides to native cyanobacteria at different rice crop stages in wetland paddy field

Biocides (herbicides and insecticides) are intensively being used in rice cultivation, despite several adverse non-target effects on native cyanobacteria. Comparative effects of three herbicides (benthiocarb, butachlor, and 2,4-D) and four insecticides (furadon, phorate, methyl parathion, and ekalux) were studied at field-recommended doses. Field experiments of rice were carried out for three consecutive seasons in completely randomized block designs in both rainy and winter croppings at the Central Rice Research Institute, Cuttack, India. Toxicities of seven biocides individually to native cyanobacteria growing with rice crops in wetland fields were assessed for growth and acetylene reduction activity (ARA); several crop parameters of paddy were estimated concomitantly. It was observed that applications of butachlor and benthiocarb individually significantly decreased growth (15.86 and 17.50%), ARA (0.16 and 0.68%), and N-yield (0.26 and 0.85%) of the native cyanobacteria, whereas these parameters increased (24.04% in growth, 0.25% in ARA, and 0.40% in N-yield) due to the application of 2,4-D. Of the tested insecticides, furadon, phorate, and methyl parathion enhanced the growth (17.8, 10.7, and 11.7%), N2-fixation (0.10, 0.097, and 0.074%), and N-yield (0.16, 0.18, and 0.20%) of cyanobacteria, whereas ekalux had no significant effect on cyanobacteria. On the basis of cyanobacterial parameters, biocides could be recommended for rice crops in the order furadon < phorate < methyl parathion < ekalux. Applications of 2,4-D and furadon individually in comparison to the rest other biocides enhanced rice yield. These finding could help to revise integrated strategies of weed and pest management in wetland rice agriculture to save non-target soil microorganisms, particularly cyanobacteria.

Deciphering the evolutionary affiliations among bacterial strains (Pseudomonas and Frankia sp.) inhabiting same ecological niche using virtual RFLP and simulation-based approaches

To decipher an evolutionary lineage between two different but important bacterial groups, i.e., Pseudomonas strain (γ-Proteobacteria) and Frankia strain (actinobacteria) growing in the same ecological niche in and around of an actinorhizal plant Hippophae salicifolia D. Don, genetic diversity and comparative molecular phylogeny have been investigated using 16S rRNA gene sequences and computer-simulated and virtually directed restriction fragment length polymorphism (RFLP) through 10 restriction enzymes. Bayesian and coalescent analyses on the basis of 16S rRNA gene sequences suggested three major groups with close proximity between Pseudomonas and Frankia isolates. This result has been further validated based on the data observed through similarity coefficient value and computational RFLP. Principal component analysis and Mandel h and k statistical analysis also confirmed and strengthen the findings. Approximately 458 aligned sequence of all the taxa were used to decipher nucleotide diversity, polymorphism and gene flow between these taxa. Thus, our results suggest for a possible co-evolution or a heterologous gene transfer of distantly related microbial forms. Further, our study also advocate for the use of computer aided, virtual RFLP analysis as a cost effective and rapid identification tool.

Impairment of ntc A gene revealed its role in regulating iron homeostasis, ROS production and cellular phenotype under iron deficiency in cyanobacterium Anabaena sp. PCC 7120

Iron deficiency ends up into several unavoidable consequences including damaging oxidative stress in cyanobacteria. NtcA is a global nitrogen regulator controls wide range of metabolisms in addition to regulation of nitrogen metabolism. In present communication, NtcA based regulation of iron homeostasis, ROS production and cellular phenotype under iron deficiency in Anabaena 7120 has been investigated. NtcA regulates the concentration dependent iron uptake by controlling the expression of furA gene. NtcA also regulated pigment synthesis and phenotypic alterations in Anabaena 7120. A significant increase in ROS production and corresponding reduction in the activities of antioxidative enzymes (SOD, CAT, APX and GR) in CSE2 mutant strain in contrast to wild type Anabaena 7120 also suggested the possible involvement of NtcA in protection against oxidative stress in iron deficiency. NtcA has no impact on the expression of furB and furC in spite of presence of consensus NtcA binding site (NBS) and −10 boxes in their promoter. NtcA also regulates the thylakoid arrangement as well as related photosynthetic and respiration rates under iron deficiency in Anabaena 7120. Overall results suggested that NtcA regulates iron acquisition and in turn protect Anabaena cells from the damaging effects of oxidative stress induced under iron deficiency.

Interaction of turmeric (Curcuma longa L.) with beneficial microbes: a review

Curcuma longa L., commonly known as turmeric, is a rhizomatous herb of the family Zingiberaceae. It is mostly used as a spice, a coloring agent and broadly used in traditional medicine such as Ayurveda, Unani, etc., Turmeric rhizomes interact with a large numbers of rhizosphere-associated microbial species, and some enter the plant tissue and act as endophytes. Both rhizospheric and endophytic species are directly or indirectly involved in growth promotion and disease management in plants and also play an important role in the modulation of morphological growth, secondary metabolite production, curcumin content, antioxidant properties, etc. The present review focuses on the rhizobacterial and endophytic bacterial and fungal populations associated with the turmeric.

Nitric oxide ameliorates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.

In cyanobacterium Anabaena 7120, iron deficiency leads to oxidative stress with unavoidable consequences. Nitric oxide reduces pigment damage and supported the growth of Anabaena 7120 in iron-deficient conditions. Elevation in nitric oxide accumulation and reduced superoxide radical production justified the role of nitric oxide in alleviating oxidative stress in iron deficiency. Increased activities of antioxidative enzymes and higher levels of ROS scavengers (ascorbate, glutathione and thiol) in iron deficiency were also observed in the presence of nitric oxide. Nitric oxide also supported the membrane integrity of Anabaena cells and reduces protein and DNA damage caused by oxidative stress induced by iron deficiency. Results suggested that nitric oxide alleviates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.