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SummaryMgtC is required for intramacrophage replication of intracellular pathogens and growth in low Mg 2+ medium. A link between these two phenotypes has been proposed due to putative Mg 2+ deprivation inside phagosome. MgtC is part of a family of proteins that share a conserved N-terminal transmembrane domain and a variable C-terminal domain. A combination of predictive and experimental approaches indicates that the Salmonella MgtC C-terminal domain is cytoplasmic, adopts a fold also found in metal transporters and RNA interacting domain, and does not bind Mg 2+ . MgtC homologues from diverse g-proteobacteria, including the extracellular pathogens Yersinia pestis, Photorhabdus luminescens and Pseudomonas aeruginosa, have been expressed in a Salmonella DmgtC strain. The Y. pestis MgtC fully replaced the Salmonella MgtC whereas P. luminescens or P. aeruginosa MgtC complemented only in low Mg 2+ medium, thus dissociating for the first time the two MgtC-related phenotypes. In addition, we identified single amino acids changes that prevent or promote MgtC role in macrophages without affecting MgtC role in low Mg 2+ culture. A Salmonella DmgtC strain showed elongated and autoaggregated bacteria in low Mg 2+ medium but not in macrophages. Taken together our results suggest that MgtC has a dual role when bacteria localize in macrophages or low Mg 2+ environment.

Selection of resistance in Spodoptera exigua (Hübner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryICresistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 g/g), whereas the 50% lethal concentration was 4.30 g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K d , and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

The role played by ␣-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that ␣-helix 4 plays a crucial role in the mechanism of pore formation.Bacillus thuringiensis is the most extensively used commercial biopesticide worldwide and is presently the sole source of toxin genes for the development of insect-resistant transgenic plants (13,15,42). The insecticidal activity of B. thuringiensis is primarily associated with its ability to synthesize a crystalline parasporal inclusion body containing highly specific insecticidal proteins (22,36). The mode of action of these insecticides involves solubilization of the crystal in the highly alkaline lepidopteran midgut lumen, activation of the toxins by intestinal proteases, recognition of one or more binding sites on the midgut brush border membrane surface followed by pore formation, and cell lysis leading ultimately to insect death (36).The elucidation by X-ray diffraction analysis of the threedimensional structures of the activated Cry1Aa (21), Cry2Aa (31), Cry3Aa (28), and Cry3Bb (16) toxins has revealed a common three-domain folding pattern. Domain I is made of seven ␣-helices, and domains II and III are composed mostly of ␤-sheets. While domain I is considered to be responsible for pore formation (37, 43, 44), domains II and III are involved in receptor binding and host specificity (11,12,24,47). Domain III is also thought to play a role in protein stability (28). The domains of the activated toxins were shown to interact with each other to yield their overall toxic effect (33, 34). Exchanging domain I from different toxins can affect crystal formation, stability, pore formation, and membrane permeability as well as the size of the pores and toxicity.The toxin is thought to form pores in the cell membrane by first inserting a hairpin composed of the hydrophobic ␣5 and the amphipathic ␣4 h...