Anumeha Singh

Attenuation of metal toxicity by frankial siderophores

In order to investigate the role of frankial siderophores in minimizing metal toxicity, Frankia strains were grown at different concentrations of Mg2+, Cu2+, and Zn2+. Growth was observed up to 500 µmol L−1 Mg2+, 10 µmol L−1 Cu2+, and 10 µmol L−1 Zn2+ while the maxima were at 200 µmol L−1 Mg2+, 1 µmol L−1 Cu2+, and 1 µmol L−1 Zn2+. The siderophore production was increased up to 500 µmol L−1 Mg2+, 10 µmol L−1 Cu2+, 100 µmol L−1 (hydroxamate type), and 200 µmol L−1 (catecholate type) Zn2+ while maximum production was found at Mg2+ (200 µmol L−1), Cu2+ (10 µmol L−1), and Zn2+ (10 µmol L−1). The results suggested that the growth was protected at higher concentrations of Mg2+ (up to 500 µmol L−1), Cu2+ (10 µmol L−1), and Zn2+ (10 µmol L−1), possibly due to enhanced siderophore production. Thus, siderophores minimize the metal-induced inhibition of growth in Frankia, likely due to regulation of nutritional imbalances and metabolic processes during adaptation towards metal stress and/or metal toxicity.

Enzymic activity of whole cells entrapped in reversed micelles. Studies on alpha-amylase and invertase in the entrapped yeast cells.

Studies have been conducted on the enzymic activity of Baker’s yeast and also of Brewer’s yeast entrapped into the reversed micelles formed by cetyl pyridinium chloride (CPC1) in n-hexane. The activities of α-amylase and invertase enzymes in the entrapped cells have been estimated and compared with those in the control experiments where there was no entrapment. The following significant observations have been made: 1. except for invertase, enzymes in Brewer’s yeast, the entrapped yeast cells showed enhanced enzymic activities; 2. when the yeast cells were entrapped inside the reversed micelles along with substrates of the two enzymes, α-amylase, and invertase, the activity of each of these enzymes showed a further enhancement in comparison to that showed in the experiments in which substrates of the individual enzymes alone were entrapped-the phenomenon of synergism; 3. when the yeast cells and the respective substrates were entrapped inside separate reversed micelles and the solutions containing entrapped cells and entrapped substrates were mixed, the activities of the individual enzymes, α-amylase and invertase, showed further enhancement in comparison to the case in which the cells and the substrates were entrapped inside the same reversed micelle (in this case also the phenomenon of synergism was observed); and (4) In the case of experiments in which there was no entrapment, it was observed that the presence of substrates induced more release of enzymes from the yeast cells.These observations on yeast cells, which to the best of our knowledge have not been reported before, should be biotechnologically relevant.

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.

Salt stress–induced changes in antioxidative defense system and proteome profiles of salt-tolerant and sensitive Frankia strains

ABSTRACT An appreciation of comparative microbial survival is most easily done while evaluating their adaptive strategies during stress. In the present experiment, antioxidative and whole cell proteome variations based on spectrophotometric analysis and SDS-PAGE and 2-dimensional gel electrophoresis have been analysed among salt-tolerant and salt-sensitive Frankia strains. This is the first report of proteomic basis underlying salt tolerance in these newly isolated Frankia strains from Hippophae salicifolia D. Don. Salt-tolerant strain HsIi10 shows higher increment in the contents of superoxide dismutase, catalase and ascorbate peroxidase as compared to salt-sensitive strain HsIi8. Differential 2-DGE profile has revealed differential profiles for salt-tolerant and salt-sensitive strains. Proteomic confirmation of salt tolerance in the strains with inbuilt efficiency of thriving in nitrogen-deficient locales is a definite advantage for these microbes. This would be equally beneficial for improvement of soil nitrogen status. Efficient protein regulation in HsIi10 suggests further exploration for its potential use as biofertilizer in saline soils.

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.

Phenotypic expression of ammonia-excreting mutants of Anabaena 7120 under nitrogen limitation

Mutants of Anabaena 7120 defective in glutamine synthetase (GS) activity were isolated following transposon mutagenesis. Mutants M11, M55 and M73 showed about 60% less GS activity in N2-grown aerobic cultures than the wild-type strain and were resistant to the glutamate analogue l-methionine-dl-sulphoximine (MSX). These mutants had the capacity to excrete N2-fixed ammonia continuously into the culture medium and showed an enhanced level of aerobic nitrogenase activity. The intracellular ammonium pool generated in N2-grown cells of mutants was found to be less than that of the wild-type strain. Similarly, ammonium uptake by these mutants was 50% less in mutants compared to the wild-type, suggesting a possible role of GS in controlling this function.

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.

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.

Phylogeny and evolutionary genetics of Frankia strains based on 16S rRNA and nifD-K gene sequences.

16S rRNA and nifD–nifK sequences were used to study the molecular phylogeny and evolutionary genetics of Frankia strains isolated from Hippöphae salicifolia D. Don growing at different altitudes (ecologically classified as riverside and hillside isolates) of the Eastern Himalayan region of North Sikkim, India. Genetic information for the small subunit rRNA (16S rRNA) revealed that the riverside Frankia isolates markedly differed from the hillside isolates suggesting that the riverside isolates are genetically compact. Further, for enhanced resolutions, the partial sequence of nifD (3′ end), nifK (5′ end) and nifD–K IGS region have been investigated. The sequences obtained, failed to separate riverside isolates and hillside isolates, thus suggesting a possible role of genetic transfer events either from hillside to riverside or vice versa. The evolutionary genetic analyses using evogenomic extrapolations of gene sequence data obtained from 16S rRNA and nifD–K provided differing equations with the pace of evolution being more appropriately, intermediate. Values of recombination frequency (R), nucleotide diversity per site (Pi), and DNA divergence estimates supported the existence of an intermixed zone where spatial isolations occurred in sync with the temporal estimates. J. Basic Microbiol. 2015, 54, 1–9