Arun Kumar Mishra

Toxicity of three herbicides to some nitrogen-fixing cyanobacteria

The effects of some common rice field herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), Machete, and Saturn, on the paddy field nitrogen-fixing cyanobacteria Nostoc linckia, Nostoc calcicola, Nostoc sp., and Anabaena doliolum were studied. These cyanobacteria were found to be more tolerant to 2,4-D (lethal doses 1500-2000 micrograms/ml) than to Machete and Saturn (lethal doses 6-8 micrograms/ml). The effects of these three herbicides on some physiological processes of N. linckia were studied. The 2,4-D stimulated the growth and nitrogen fixation up to 100 micrograms/ml concentration (a dose higher than the field dose, i.e., about 40 micrograms/ml), recommended for field application. However, with Machete and Saturn this type of stimulation was not observed even at lower concentrations. Similarly, the uptake of nutrients, such as NO3- and NH+4, was also inhibited by Machete and Saturn. However, 100 micrograms/ml 2,4-D stimulated the uptake of NO3- but not of NH+4; higher doses of 2,4-D inhibited the uptake of both nutrients. Factors such as pH, organic carbon sources (glucose and acetate), and amino acids were found to regulate the toxicity of all three herbicides to N. linckia. Lower pH enhanced the toxicity of all three herbicides, whereas higher pH (up to 9.0) lowered it. Glucose and acetate (each 500 micrograms/ml) protected against the toxicity of 2,4-D and Saturn, but not against Machete. Whereas glutamine, arginine, serine, and tryptophan conferred upon N. linckia a greater protection against the toxicity of all three herbicides, methionine did not do so, and the presence of methionine with herbicide in the culture medium resulted in greater toxicity to N. linckia than that in the presence of the herbicide alone.

Molecular phylogeny, population genetics, and evolution of heterocystous cyanobacteria using nifH gene sequences

In order to assess phylogeny, population genetics, and approximation of future course of cyanobacterial evolution based on nifH gene sequences, 41 heterocystous cyanobacterial strains collected from all over India have been used in the present study. NifH gene sequence analysis data confirm that the heterocystous cyanobacteria are monophyletic while the stigonematales show polyphyletic origin with grave intermixing. Further, analysis of nifH gene sequence data using intricate mathematical extrapolations revealed that the nucleotide diversity and recombination frequency is much greater in Nostocales than the Stigonematales. Similarly, DNA divergence studies showed significant values of divergence with greater gene conversion tracts in the unbranched (Nostocales) than the branched (Stigonematales) strains. Our data strongly support the origin of true branching cyanobacterial strains from the unbranched strains.

Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Phylogenetic comparison among the heterocystous cyanobacteria based on a polyphasic approach

Phylogenetic comparison has been done among the selected heterocystous cyanobacteria belonging to the sections IV and V. The hierarchical cluster analysis based on antibiotics sensitivity showed a distant relationship between the members of Nostocales and Stigonematales. Thus, multiple antibiotic resistance pattern used as marker provide easy, fast, and reliable method for strain discrimination and genetic variability. However, morphological, physiological (both based on principal component analysis) and biochemical analysis grouped true branching cyanobacteria along with the members of section IV. Molecular analysis based on 16S rRNA gene sequences revealed that Hapalosiphon welwitschii and Westiellopsis sp. were grouped in cluster I whereas Scytonema bohnerii, a false branching genera showed a close proximity with Calothrix brevissima in cluster II. Cluster III of clade 2 included Nostoc calcicola and Anabaena oryzae which proved the heterogeneity at the generic level. Cluster IV the largest group of clade 2 based on 16S rRNA gene sequences includes six strains of the genera Nostoc, Anabaena, and Cylindrospermum showing ambiguous evolutionary relationship. In cluster IV, Anabaena sp. and Anabaena doliolum were phylogenetically linked by sharing 99% sequence similarity. Probably, they were of the same genetic makeup but appear differently under the diverse physiological conditions. Section IV showed polyphyletic origin whereas section V showed monophyletic origin. Results suggested that either morphological or physiological or biochemical or molecular attribute is not sufficient to provide true diversity and phylogeny of the cyanobacteria at the generic level and thus, a polyphasic approach would be more appropriate and reliable.

Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data

Taxonomic affiliations and molecular diversity of 41 heterocystous cyanobacteria representing 12 genera have been assessed on an evolutionary landscape using rbcl gene sequence data-based phylogenomics and evogenomics approaches. Phylogenetic affiliations have clearly demonstrated the polyphyly of the true branching cyanobacteria, along with a frequent intermixing amongst the heterocystous cyanobacteria. The monophyletic origin of the heterocystous cyanobacteria was also quite evident from maximum parsimony and neighbor joining analyses. Incongruency with the traditional scheme of cyanobacterial taxonomy was frequently observed, thus advocating towards some re-amendments in the cyanobacterial classificatory schemes. Evogenomics analyses of gene sequence data gave a clear indication about the greater evolutionary pace of the unbranched cyanobacteria as compared to the branched forms. It was evident that the order Nostocales would be controlling the future pace of evolution of heterocystous cyanobacteria. The cyanobacteria Nostoc was found to have the greatest genetic heterogeneity amongst the studied genera, along with some evidence towards events of lateral gene transfer amongst the heterocystous cyanobacteria in case of the rbcl gene. Thus, heterocystous cyanobacteria were found to be a fast evolving group, with estimates of gene conversion tracts pointing towards the unbranched heterocystous cyanobacteria being at the base of evolutionary diversifications of the complete heterocystous lineage.

Chemotaxonomy of heterocystous cyanobacteria using FAME profiling as species markers

The fatty acid methyl ester (FAME) analysis of the 12 heterocystous cyanobacterial strains showed different fatty acid profiling based on the presence/absence and the percentage of 13 different types of fatty acids. The major fatty acids viz. palmitic acid (16:0), hexadecadienoic acid (16:2), stearic acid (18:0), oleic acid (18:1), linoleic (18:2), and linolenic acid (18:3) were present among all the strains except Cylindrospermum musicola where oleic acid (18:1) was absent. All the strains showed high levels of polyunsaturated fatty acid (PUFAs; 41–68.35%) followed by saturated fatty acid (SAFAs; 1.82–40.66%) and monounsaturated fatty acid (0.85–24.98%). Highest percentage of PUFAs and essential fatty acid (linolenic acid; 18:3) was reported in Scytonema bohnerii which can be used as fatty acid supplement in medical and biotechnological purpose. The cluster analysis based on FAME profiling suggests the presence of two distinct clusters with Euclidean distance ranging from 0 to 25. S. bohnerii of cluster I was distantly related to the other strains of cluster II. The genotypes of cluster II were further divided into two subclusters, i.e., IIa with C. musicola showing great divergence with the other genotypes of IIb which was further subdivided into two groups. Subsubcluster IIb1 was represented by a genotype, Anabaena sp. whereas subsubcluster IIb2 was distinguished by two groups, i.e., one group having significant similarity among their three genotypes showed distant relation with the other group having closely related six genotypes. To test the validity of the fatty acid profiles as a marker, cluster analysis has also been generated on the basis of morphological attributes. Our results suggest that FAME profiling might be used as species markers in the study of polyphasic approach based taxonomy and phylogenetic relationship.

Decoding cyanobacterial phylogeny and molecular evolution using an evonumeric approach.

Forty-one heterocystous cyanobacteria strains representing 12 cyanobacterial genera collected from all across India were assessed in phylogenetic and evolutionary perspectives. The structural gene 16S ribosomal RNA (rRNA) and the functional genes nifD and psbA were selected as molecular chronometers in this study. The phylogenetic analyses demonstrated the monophyly of heterocystous cyanobacteria with significant intermixing, along with establishing the polyphyly of Stigonematales, strongly supporting the need for re-amendments in cyanobacterial taxonomy and systematics. Molecular trends obtained did not clearly reflect the phenotypic affiliations, thus advocating for genetic characterizations using more molecular markers. Large-scale evonumeric extrapolations of gene sequence data of all the three molecular markers was performed to assess the evolutionary pace of heterocystous cyanobacteria on the basis of nucleotide diversity, recombination frequencies, and the DNA divergence between the sampled taxa. The obtained results tilted the evolutionary pace in favor of the less complex Nostocales thus indicating that possibly the simple non-branched forms are more flexible and adaptive for evolutionary diversifications as compared to the more complex and branched ones. This study hence represents a unique blend of molecular phylogeny with evogenomic sequence analyses for understanding the genetic diversity, phylogeny, and evolutionary pace within the heterocystous cyanobacteria.

Regulation of calcium ion and its effect on growth and developmental behavior in wild type and ntcA mutant of Anabaena sp. PCC 7120 under varied levels of CaCl2

A study of calcium ion regulation in Anabaena 7120 and its derivative mutant (CSE2) strain impaired in ntcA gene were investigated in terms of altered morphological and physiological responses against various levels of calcium stress (0–100 mM). Calcium concentration of 10 mM was found to be inhibitory while 100 mM proved lethal for both wild type and mutant strain. The involvement of Ca2+ in the regulation of cellular processes has been described in terms of an influx or efflux of Ca2+ from the cytosol. A biphasic calcium uptake with difference in calcium influx and efflux rate was responsible for differential amount of remaining calcium which followed a decreasing trend both for wild type and mutant. Low Ks 0.5 and high Vmax in mutant suggest heavy and less restricted influx of calcium ion. Further, the interactive effect of calcium influx/efflux rate, remaining Ca2+ and intracellular levels of Na+ and K+ may be attributed for the degree of membrane damage and growth sustenance during exogenous supply of calcium salt. Widening in heterocyst spacing pattern, decreased heterocyst frequency and formation of abnormal cell structures at higher concentration (100 mM CaCl2) suggest that calcium mediated regulatory process modulate heterocyst frequency and maintenance of cell structure. Further, poor regulation of calcium ion homeostasis in ntcA suggests that the calcium level and ntcA gene expression are inter-related.

Physiological interactions in Azolla-Anabaena system adapting to the salt stress

Abstract The effect of the different levels of salinity on some important physiological parameters among six species of Azolla has been investigated. Salinity reduced the growth (dry weight) and nitrogenase activity in Azolla-Anabaena association while heterocyst frequency was sharply increased. Minimum sodium accumulation and electrolyte leakage (EL) was observed up to 20mMNaCl whereas nitrate was accumulated significantly. Beyond 20mMNaCl, fronds having higher amount of Na+ and EL inhibited the accumulation and reduction of nitrate suggests that 20mMNaCl is the critical concentration. Highest superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline content might be the probable reason for the low level of EL, malondialdehyde (MDA) content and Na+ accumulation in Azolla species. Lower Na+ accumulation, solute leakage and lipid peroxidation along with the higher nitrogen demand and the proline accumulation with a high ability of oxygen radical scavenging in A. microphylla might be the reasons to grow at higher NaCl concentrations.

Sodium transport and mechanism(s) of sodium tolerance in Frankia strains

The mechanism(s) underlying differential salt sensitivity/tolerance were investigated in the terms of altered morphological and physiological responses against salinity such as growth, electrolyte leakage, Na+ uptake, efflux, accumulation and intracellular concentrations of macronutrients among the Frankia strains newly isolated from Hippöphae salicifolia D. Don. Growth was minimally reduced at 500 and 250 mM NaCl respectively in HsIi10 and rest of the strains (HsIi2, HsIi8, HsIi9) which proved that 500 and 250 mM NaCl are the critical concentrations for the respective strains. The differences in the sodium influx/efflux rate was responsible for the differential amount of remaining sodium among the frankial strains and might be one of the primary determinants for the reestablishment of macronutrients (Mg2+, Ca2+ and K+) during salinity. Secondly, the interactive effect of sodium influx/efflux rate, remaining sodium and intracellular macronutrients (Mg2+, Ca2+ and K+) concentration has been responsible for the extent of membrane damage and growth sustenance of the tolerant/sensitive frankial strains during salinity. HsIi10 showed better co‐regulation of various factors and managed to tolerate salt stress up to considerable extent. Therefore, HsIi10 can serve as a potential biofertilizer in the saline soil.