Tunicamycin-resistant mutants of Bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis

Mutants of Bacillus amyloliquefaciens resistant to at least 10 micrograms/ml of tunicamycin were isolated and shown to be pleiotropic. The mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin G and tetracycline. They were more autolytic, presumably due to an altered cell wall. The mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporulation frequency and being more motile.     

Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annum</Emphasis> L.)

Plants, when exposed to abiotic or biotic stress, produce several pathogenesis-related proteins to counteract the effects of stress. Osmotin is one of the important pathogenesis-related proteins induced during several stress conditions. We have developed improved salt stress tolerant transgenic chilli pepper plants (Capsicum annum L. var. Aiswarya 2103) by ectopic expression of the Nicotiana tabaccum osmotin gene using Agrobacterium tumefaciens EHA105 as a vector. Four-week-old chilli pepper leaves were used as an explant and A. tumefaciens EHA105 harboring pBINASCOSM plasmid that contains osmotin gene under the control of CaMV 35S promoter and npt II as a selectable marker was used in co-cultivation. Transgene integration and expression were analyzed using molecular, immunochemical, and biochemical assays. PCR and Southern blot analysis confirmed that osmotin gene has been successfully integrated into the genome of chilli pepper plants. The osmotin gene was stably segregated and expressed in T₂ generation transgenic chilli pepper plants, and it was confirmed by Western blot analysis. Biochemical assays of these putative transgenic plants revealed enhanced levels of chlorophyll, proline, glycinebetaine, APX, SOD, DHAR, MDHAR, GR, and relative water content. Yield potential of the putative transgenic chilli pepper plants was evaluated under salinity stress conditions in a green house. The putative transgenic chilli pepper plants overexpressing the osmotin gene were morphologically similar to wild-type plants and produced 3.32 kg chilli pepper fruits per plant at 300 mM NaCl concentration.     

Starch-degrading enzymes of two species ofFusarium

Starch supported production of maximum α-amylases (dextrinizing and saccharifying) byFusarium oxysporum andF. scirpi. Addition of gibberellic acid resulted in an increased production of α-amylase. Presence of glucose depressed the enzyme production. pH 4.5 and 4.0 was found to be optimum for the dextrinizing enzyme secreted by both species. The temperature of 25 and 40 °C was optimum for the dextrinizing enzyme secreted byF. oxysporum andF. scirpi, respectively. Saccharifying enzymes of both species showed their optimum at pH 6.9. The optimum temperature for the activity of the saccharifying enzyme was 30 and 40 °C, respectively.     

Overproduction and rapid purification of the phosphoenolpyruvate:sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli.

We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures.     

Pattern of protein profiles of reproductive organs after induced bilateral cryptorchidism in albino rats

Bilateral cryptorchidism was induced surgically in albino rats and pattern of protein profiles was studied in reproductive organs. Cryptorchidism activated tissue proteolysis leading to overall degradation in soluble and structural protein fractions and in amino acids leading to prevalence of negative nitrogen balance in the reproductive organs. The testicular hypoalbuminic and hypoglobulinic conditions seem to be responsible for oligo-astheno-spermia associated with cryptorchidism.     

Immobilization of amyloglucosidase on polystyrene anion exchangeresin II. Kinetics and stabilities

Summary Amyloglucosidase (exo-1, 4- -D-glucosidase, EC 3.2.1.3), was coupled to glutaraldehyde activated Indion 48-R (a cross-linked macroporous anion exchanger) by Schiff base reaction. Immobilization brought about a marginal increase in the apparent K_m. The bound enzyme exhibited increased stability towards urea and metal ions, but was less stable in the presence of guanidine hydrochloride. Immobilized amyloglucosidase could be stored at 4°C (in wet state) for 6–8 months without any apparent loss of activity.     

Calcium-Regulated in Vivo Protein Phosphorylation in Zea mays L. Root Tips

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with ³²P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent protein kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.     

An enzymic method for the determination of chitin and chitosan in fungal cell walls

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain ofNeurospora crassa. Chitinase, obtained from cultures ofSerratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.     

Inactivation of clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase by sodium nitrite.

Clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase activity was investigated after in vitro or in vivo treatment with sodium nitrite. In vitro treatment of commercially available Clostridium pasteurianum ferredoxin with sodium nitrite inhibited ferredoxin activity. Inhibition of ferredoxin activity increased with increasing levels of sodium nitrite. Ferredoxin was isolated from normal C. pasteurianum and Clostridium botulinum cultures and from cultures incubated with 1,000 micrograms of sodium nitrite per ml for 45 min. The activity of in vivo nitrite-treated ferredoxin was decreased compared with that of control ferredoxin. Pyruvate-ferredoxin oxidoreductase isolated from C. botulinum cultures incubated with 1,000 micrograms of sodium nitrite per ml showed less activity than did control oxidoreductase. It is concluded that the antibotulinal activity of nitrite is due at least in part to inactivation of ferredoxin and pyruvate-ferredoxin oxidoreductase.     

Characterization of genomic DNA of mycobacteriophage I3

The GC content of mycobacteriophage I3 DNA is 67% as determined from thermal melting analysis, buoyant density in CsCl gradient, and 5-deoxymononucleotide analysis. High-resolution melting of I3 DNA revealed that the base distribution is random. Studies with methylation-specific restriction enzymes and high voltage electrophoretic analysis of 5-deoxymononucleotides did not indicate the presence of any unusual or methylated bases in I3 DNA. The molecular size of I3 DNA is estimated to be about 135 Kbp, based on the restriction fragment size distributions and sedimentation in sucrose gradients. The restriction cleavage pattern by a variety of restriction endonucleases has been determined. The circularly permuted nature of I3 DNA, indicated from the restriction patterns, has been confirmed by Southern blot hybridizations and 5 end group analysis.