Soft rot inciting <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> (syn. <Emphasis Type="Italic">Erwinia carotovora</Emphasis>) is unlikely to be transmitted as a latent pathogen in micropropagated banana

This study was undertaken to ascertain if the soft rot inciting Pectobacterium carotovorum/Erwinia carotovora would pass through the micropropagated bananas as a latent pathogen and cause disease during or post acclimatization. In vitro cultures of ‘Grand Naine’ were exposed to the pathogen by providing 100 μl of inoculum (0.001–1.0 at OD₆₀₀ nm) at the lower leaf axil. These cultures showed a gradual development of soft rot symptoms coupled with obvious bacterial colony growth on banana proliferation medium and consequent plant mortality within a month irrespective of the inoculum level employed. Plants carried forward to acclimatization following inoculation in vitro failed to establish ex vitro. Monitoring the normal field-grown suckers at culture initiation through PCR screening employing soft rot Erwinia primers did not show the amplification of the 119-bp fragment as seen with the pure cultures of pathogen. Further testing of micropropagated banana plants through soil inoculation, in vitro culturing and PCR screening ruled out the possibility of the pathogen surviving in micropropagated stocks in latent form as the organism outgrew and killed the cultures. It emerged that the infection possibly takes place in the nursery. This information will be of particular value for the plant tissue culture industry, plant pathologists and quarantine agencies.     

Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met₁₀₁ and Met₁₃₃, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met₁₀₁ and Met₁₃₃ residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Mating compatibility among helicoverpa armigera (Lepidoptera: Noctuidae) occurring on selected host plants and Bt cotton survivors

Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae) is a well-known polyphagous insect pest. Mating compatibility among the insects occurring on different host plants is essential for free gene flow among populations. We tested the extent of crossability and fecundity of the insects that survived on Bacillus thuringiensis (Bt) cotton with those occurring on pigeon pea, Cajanus cajun (L.) Millsp., non-Bt cotton, Gossypium hirsutum L.; sunflower, Helianthus annuus; sorghum, Sorghum bicolor L. Moench.; okra, Abelmoschus moschatus Medikus; chickpea, Cicer arietinum L.; marigold, Tagetes spp.; and tomato, Lycopersicum esculentum L., crops. The insects from different crops were freely crossable with those collected from Bt cotton and among themselves. The average fecundity across different crosses ranged from 314.1 to 426.3 in direct and from 305.8 to 421.7 eggs per female in reciprocal crosses. In any given cross, a minimum of 85.89% egg hatch was recorded. Furthermore, the F1 individuals of different cross combinations were found to cross freely with their parents (BC1) and among themselves with similar fecundity and egg hatch. High crossability among H. armigera occurring on different host plants suggests that crop mosaics that may exist in countries such as India could play an important role as natural, nonstructured refugia and prolong the durability of the genes deployed for controlling this insect.     

Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera,Culicidae) larvae

Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified. Other Cry4Ba binding proteins identified include the putative lipid raft proteins flotillin and prohibitin, V-ATPase B subunit and actin. These identified proteins might play important roles in mediating the toxicity of Cry4Ba due to their location in the gut brush border membrane. Cadherin-type protein was not identified, although previously, we identified a midgut cadherin AgCad1 as a putative Cry4Ba receptor in Anopheles gambiae mosquito larvae [Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101–5110]. Other identified proteins in this study that might have lesser roles include mitochondrial proteins such as ATP synthase subunits, mitochondrial processing peptidase and porin; which are likely contaminants from mitochondria and are not brush border membrane components. Trypsin-like serine protease was also identified as a protein that binds Cry4Ba. Identification of these toxin-binding proteins will lead to a better understanding of the mode of action of this toxin in mosquito.     

Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli

A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P(i) bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P(i). In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-anomeric hydroxyl group of D-glucose is highly required. However, even when it is removed enzyme activity remains. The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted. The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.