Increased expression of the insect control protein genes ofBacilus thunrngiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modIfied (3% nucleotide difference) and to fully modified (21% nucleotide difference) crylA(b) and crylA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified crylA(b) gene had a 10-fold higher level ofinsect control protein and plants with the fully modified crylA(b) had a 100-fold higher level of CryIA Insect control proteins from a prokaryotic source, Bacillus thuringiensis var. kurstaki (B.t.k.; ref. 1) are specific for lepidopteran insects and exhibit no activity against humans, other vertebrates, and beneficial insects (2). These properties have made the genes of these insect-specific proteins attractive candidates for genetic modification of crops for protection against lepidopteran pests. Genes encoding lepidopteran-specific insect control proteins have been cloned and sequenced. Truncated genes, which produce insecticidally active protein, have been expressed in tomato (3), tobacco (4), and cotton (5). Field tests of these plants revealed that higher levels of insect control protein in the plant tissue would be required to obtain commercially useful plants (6).The insect control proteins are highly expressed in their natural host, B. thuringiensis. Up to 50% of the total protein in sporulated cultures ofB.t.k. are the insect control proteins deposited as crystals within the cell. Insect control protein genes are expressed well in Escherichia coli (7) or Pseudomonas (8). Poor expression in plants is a well-reported characteristic of the B.t.k. insect control proteins. Truncating the gene, keeping essentially the N-terminal half of the protein intact, results in improved expression of the gene in plants to barely detectable levels (3, 4). The use ofdifferent promoters, fusion proteins, and leader sequences has not significantly increased insect control protein gene expression (4, 9).We hypothesized that a gene with a sequence adapted for a Gram-positive prokaryote may not be the appropriate coding sequence for efficient plant expression. Examination of the insect control protein gene coding sequence indicated that it differs significantly from plant genes in G+C content. Multiple sequences motifs that are not common in the coding region of plant genes were found to be common in the wild-type (WT) crylA(b) sequence. These included localized regions of A+T richness resembling plant introns (10), potential plant polyadenylylation signal sequences (11), ATTTA sequences, which have been shown to destabilize mRNA in other systems (12), and codons rarely used in plants...

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlAS81F and GrlAS81Y quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg2+ bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg2+ concentrations to support DNA cleavage by GrlAS81F topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the ternary enzyme-quinolone-DNA complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg2+ bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically-relevant quinolone resistance mutations in bacterial type II topoisomerases.

Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlAE85K, GrlAS81F/E85K, GrlAE85A, GrlAS81F/E85A and GrlAS81F Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg2+ concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein–quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.