Satya Shila Singh

MTHFR C677T and A1298C polymorphisms are risk factors for Down’s syndrome in Indian mothers

AbstractDowns syndrome (DS), a chromosomal disorder due to trisomy 21, results mostly from nondisjunction in maternal meiosis. The present case-control study examined the association of genetic polymorphisms with predisposition to nondisjunction. Two common polymorphisms (SNPs), C677T and A1298C, in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene involved in folate metabolism, are known to lower the activity of this enzyme. Three hundred and fourteen mothers (with DS children and controls), mostly from the eastern states of India, were genotyped for the two above-mentioned SNPs. Significant association with both of these SNPs were detected, more specifically, in the mothers of DS children homozygous for the polymorphic alleles 677 T and 1298 C. The relative risk of T (C677T) and C (A1298C) homozygosity in mothers for DS-affected pregnancy was 7 (OR 7.67, 95% CI 1.67-35.08, P=0.003) and 4 (OR 4.40, 95% CI 1.45-13.26, P=0.008), respectively. Moreover, all 677TT mothers studied were less than 31 years of age, whereas no correlation with maternal age was observed for A1298C genotypes. Interestingly, all of the young 677TT mothers had either a first- or secondborn child with DS. Thus, this study reports that young Indian mothers with TT genotypes are genetically predisposed to nondisjunction due to abnormal folate metabolism.

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.

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.

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.

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.

Diversity of Frankia strains nodulating HippÖphae salicifolia D. Don using FAME profiling as Chemotaxonomic markers

Twelve Frankia strains isolated from HippÖphae salicifolia D. Don or Alnus glutinosa or Comptonia peregrine, showed the significant variation in fatty acid composition viz. palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidic acid (20:0) and erucic acid (22:1) suggesting the strain specific variability among the Frankia strains. Presence of Erucic acid (22:1), a major component of the oil obtained from the seeds of Brassica sp., albeit in lesser amount in the few studied frankial strains, is the first report. Cluster analysis on the basis of fatty acid composition suggests the presence of two distinct clusters with similitude coefficient ranging from 0.75 to 1.00. Cluster I with HsIi2 showed great divergence from other 11 frankial strains (Cluster II). The two sub groups were distinguished in cluster II: IIa contained five strains isolated from H. salicifolia and these strains are distantly related to the strains of cluster IIb isolated from different host. There is high degree of similarity among the frankial strains of Cluster IIb which suggests that the frankial strains might be evolved from the same ancestor. FAME profiling might be useful tool in the study of polyphasic approach based taxonomy and phylogenetic relationship. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)