Spatial distribution and community structure of microbiota associated with cowpea aphid (Aphis craccivora Koch).

Aphid populations were collected on cowpea, dolichos, redgram and black gram from Belagavi and Udupi locations. The samples were shotgun sequenced using the Illumina NovaSeq 6000 system to understand the spatial distribution and community structure of microbiota (especially bacteria) associated with aphids. In the present study, we identified obligatory nutritional symbiont Buchnera aphidicola and facultative symbionts Rickettsia sp. and Bacteroidetes endosymbiont of Geopemphigus sp. in all the aphid samples studied, although in varied abundance. On the other hand, Serratia symbiotica, Arsenophonus sp. and Acinetobacter sp. were only found in aphids on specific host plants, suggesting that host plants might influence the bacterial community structure. Furthermore, our study revealed that microbiota other than bacteria were highly insignificant in the aphid populations. Additionally, functional annotation of aphid metagenomes identified several pathways and enzymes involved in various physiological and ecological functions. Amino acid and vitamin biosynthesis-related pathways were predominant than carbohydrate metabolism, owing to their feeding habit and nutritional requirement. Chaperones related to stress tolerance such as GroEL and DnaK were identified. Enzymes involved in toxic chemical metabolisms such as glutathione transferase, phosphodiesterases and ABC transferases were observed. These enzymes may confer resistance to pesticides in the aphid populations. Overall, our results support the importance of host plants in structuring bacterial communities in aphids and show the functional roles of symbionts in aphid survival and development. Thus, these findings can be the basis for further detailed investigations and devising better strategies to manage the pests in field conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03142-1.

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine.

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

Studies on the nocodazole-induced GTPase activity of tubulin.

The tubulin dimer contains two guanine nucleotide binding sites, a nonexchangeable site occupied by GTP and an exchangeable site (E-site) occupied by GTP or GDP. Under the conditions used in this study the E-site GTP was hydrolyzed at a rate of 8 x 10(-5) s(-1) at 37 degrees C. This rate is stimulated four- to fivefold by nocodazole, an antimitotic drug. We studied the characteristics of this drug-stimulated reaction to learn more about the hydrolytic center of tubulin. The reaction, studied using single turnover kinetics, i.e., in the absence of added GTP, has a pH optimum of 6.6 to 7.2 and an activation energy of 88 kJ x mol(-1). It is first-order with respect to tubulin-GTP, indicating that hydrolysis is not dependent on an aggregation process. Divalent cations stimulate the hydrolysis three- to six-fold over the rate in the presence of EDTA. The reaction has a requirement for Na+ that is not satisfied effectively by other monovalent cations. In contrast, Na+ and K+ are almost equally effective in the tubulin assembly reaction. Different purine nucleoside triphosphates can bind to the E-site and are hydrolyzed. GTP and ITP are hydrolyzed at equivalent rates and XTP and ATP are hydrolyzed at a rate about half as fast. Hydrolysis is inhibited by the reagents diethylpyrocarbonate and N-ethylmaleimide, although the GTPase activity is less sensitive than the assembly reaction. The reaction rate in D2O is twice the rate in H2O. This inverse isotope effect suggests the involvement of a sulfhydryl group in the rate-limiting step in the reaction.

Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling.

After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus).

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.