Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.

Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki.

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.

Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation survival.

Starvation proteins synthesized by Escherichia coli at the onset of carbon starvation (R. G. Groat and A. Matin, J. Indust. Microbiol. 1:69-73, 1986) exhibited four temporal classes of synthesis in response to glucose or succinate starvation, indicating sequential expression of carbon starvation response (cst) genes. A cst mutant of E. coli showed greatly impaired carbon starvation survival. Thus, it appears that E. coli undergoes a significant molecular realignment in response to starvation, which increases its resistance to this stress. New polypeptides were also synthesized by E. coli in response to phosphate or nitrogen starvation. Some of these polypeptides were unique to a given starvation regimen, but at least 13 appeared to be synthesized regardless of the nutrient deprivation causing the starvation.

Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules.

Host plant glutamine synthetase (GS) has been purified 100-fold from N(2)-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody-containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N(2)-fixing alfalfa nodules.Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts.

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.).

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO(3) (-) or NH(4) (+). Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH(4) (+)-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.Enzymological data indicate that nodules of these ineffective alfalfa genotypes are uniquely differentiated plant organs. Decreasing or constant plant GS and GOGAT specific activities in ineffective nodules in response to applied N suggest that factors in addition to N supply are involved in the induction of high levels of plant ammonia-assimilating enzymes in nodules. Genotype differences observed for nodule enzyme specific activities support the concept that ineffectiveness may be expressed in different ways within the nodule. Senescence was evident in ineffective nodules of N-treated plants of both genotypes, indicating that nodule senescence induced by applied N may not be closely linked to symbiotic effectiveness in alfalfa. Data for ammonia-assimilating enzymes in roots suggest the GS/GOGAT pathway operates only at low levels of soil N and that GDH functions to detoxify high levels of soil NH(4) (+).

Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN.

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO(3) (-) at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO(3) (-)-N per hectare and harvested plants treated with 40 kilograms NO(3) (-)-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N(2)-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.After an initial lag of 24 hours, specific activities of alfalfa nodule glutamine synthetase, NADH-glutamate synthase, and NAD-glutamate dehydrogenase (oxidative amination) decreased similar to but less rapidly than acetylene reduction activity. Increased specific activities of these nodule enzymes occurred as acetylene reduction activity increased and shoot growth resumed. The observed rates of glutamine synthetase and glutamate synthase were sufficient to assimilate ammonia produced via symbiotic N(2) fixation. Nodule NADH-dependent glutamate dehydrogenase (reductive amination) specific activity was not associated with changes in acetylene reduction activity.The data indicate that host plant glutamine synthetase and NADH-glutamate synthase function to assimilate symbiotically fixed N and that NADH-dependent glutamate dehydrogenase may function in ammonia assimilation during senescence in alfalfa nodules.