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An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

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Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.

The separately cloned large and small subunits of AHAS isozyme III from Escherichia coli have been isolated and purified. The essentially pure small subunit (17 kDa ilvH product) was obtained by a procedure exploiting its low solubility. The large, catalytic subunit (62 kDa ilvI product) was isolated by standard techniques, to > or = 95% purity. The large subunit has low catalytic activity relative to holoenzyme (approximately 5%) but shows similar substrate specificity and qualitatively similar cofactor dependence and inhibition by a sulfonylurea herbicide. Its activity is insensitive to valine, and the protein does not bind valine. The small subunit binds valine with Kd = 0.2 mM. Reconstitution of the holoenzyme from its subunits leads to a complex with the properties of the native protein, including valine inhibition of activity with Ki = 12 microM. Reconstitution titrations confirm the 1:1 stoichiometry of subunit assembly and a tendency to dissociation (about 50% dissociation near 0.1 microM subunit). Size exclusion HPLC indicates that either subunit alone is largely monomeric, and that assembly of the holoenzyme (two large + two small subunits, 150-160 kDa) requires FAD. On the basis of its homology with pyruvate oxidase and pyruvate decarboxylase, we suggest that the active sites of AHAS III are located at the interface of a dimer of catalytic subunits. Our experiments suggest that such a dimer is not stable except in the presence of the small subunits. The association of valine with sites on the regulatory subunits presumably influences the active sites by an allosteric conformational effect.

The thiamin diphosphate (ThDP)-dependent biosynthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP ؊ ), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP ؊ from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP ؊ is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg 276 ) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound Acetohydroxyacid synthase (AHAS) 1 belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes that catalyze reactions whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1, 2). However, despite the similarity of AHASs to, for example, pyruvate decarboxylases and pyruvate oxidases (3-6), AHASs carry out a specific carboligation reaction in which the decarboxylation of pyruvate is followed by the condensation of the bound hydroxyethyl-ThDP anion/enamine (HEThDP Ϫ ) intermediate with a second aliphatic ketoacid to form an acetohydroxyacid (Fig. 1). Whereas the role of the enzyme in the first steps in AHAS catalysis (i.e. activation of ThDP (7), decarboxylation of pyruvate, and formation of HEThDP Ϫ (step 1 in Fig. 1)) is comparable with the function of other members of its homologous family (8), it has been difficult to suggest roles for specific protein residues in the final steps (2 and 3) of the reaction in which the product acetohydroxyacid is formed and released.One reason for this uncertainty has been the lack of clear direct information on the structure of the regions of the active site that might be involved in selective reaction of HEThDP Ϫ with a second ketoacid. Although we have proposed a homology model for AHAS isozyme II from Escherichia coli (9), based on the crystal structure of pyruvate oxidase from Lactobacillus plantarum (LpPOX) (4), these two proteins have very different sequences in the region that is likely to interact with the second substrate. In the first published crystal structure of an AHAS, that of the catalytic subunits of the yeast enzyme (10), this region was disordered. The recent publication of a new structure of the yeast enzyme with a tightly bound herbicide (11) now provides a solid framework for consideration of the role of the protein in directing the fate of the HEThDP Ϫ intermediate. A second, equally serious obstacle to the understanding of the mechanism of AHAS has been a lack of experimental tools for studying the rates of individual steps in the reaction ...

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

The microbial biota dwelling in the mucus, on the surface, and in the tissues of many coral species may have an important role in holobiont physiology and health. This microbiota differs with coral species, water depth, and geographic location. Here we compare the surface mucus microbiota of the coral Fungia granulosa from the natural environment with that from individuals maintained in aquaria. Molecular analysis revealed that the microbial community of the mucus microlayer of the coral F. granulosa includes a wide range of bacteria and that these change with environment. Coral mucus from the natural environment contained a significantly higher diversity of microorganisms than did mucus from corals maintained in the closed-system aquaria. A microbial community shift, with the loss of several groups, including actinobacterial and cyanobacterial groups, was observed in corals maintained in aquaria. The most abundant bacterial class in F. granulosa mucus was the Alphaproteobacteria, regardless of whether the corals were from aquaria or freshly collected from their natural environment. A significantly higher percentage of bacteria from the Betaproteobacteria class was evident in aquarium corals (24%) when compared with corals from the natural environment (3%). The differences in mucus-inhabiting microbial communities between corals from captive and natural environments suggest an adaptation of the mucus bacterial communities to the different conditions.