To help explain a role of the Shiga toxin family in hemorrhagic colitis and hemolytic-uremic syndrome in humans, it has been hypothesized that these toxins cause direct damage to the vascular endothelium. We now report that Shiga toxin purified from Shigella dysenteriae 1 does indeed have a direct cytotoxic effec on vascular endothelial cells in cultures. Human umbilical vein endothelial cells (HUVEC) in confluent monolayers were reduced 50% by 10-8 M Shiga toxin after a lag period of 48 to 96 h. In comparison, nonconfluent HUVEC were reduced 50% by 10-10 M Shiga toxin within a 24-h period. These data suggest that dividing endothelial cells are more sensitive to Shiga toxin than are quiescent cells in confluent monolayers. Both confluent and nonconfluent HUVEC specifically bound '25I-Shiga toxin. However, in response to the toxin, rates of incorporation of [3H]leucine into protein were more severely reduced in nonconfluent cells than in confluent cells. Toxin inhibition of protein synthesis preceded detachment of cells from the substratum. The specific binding of '251-Shiga toxin to human endothelial cells and the cytotoxic response were both toxin dose dependent and neutralized by anti-Shiga toxin antibody. Heat-denatured Shiga toxin was without the cytotoxic effect. In addition, the complete culture system contained less than 0.1 ng of bacterial endotoxin per ml, as measured by the Limulus amoebocyte lysate test.

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Bacillus subtilis has duplicate isochorismate synthase genes, menF and dhbC. Isochorismate synthase is involved in the biosynthesis of both the respiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB). Several menF and dhbC deletion mutants were constructed to identify the contribution made by each gene product to MK and DHB biosynthesis. menF deletion mutants were able to produce wild-type levels of MK and DHB, suggesting that the dhbC gene product is able to compensate for the lack of MenF. However, a dhbC deletion mutant produced wild-type levels of MK but was DHB deficient, indicating that MenF is unable to compensate for the lack of DhbC. A menF dhbC double-deletion mutant was both MK and DHB deficient. Transcription analysis showed that expression of dhbC, but not of menF, is regulated by iron concentration. A dhbA::lacZ fusion strain was constructed to examine the effects of mutations to the iron box sequence within the dhb promoter region. These mutations abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in B. subtilis.Isochorismate synthase is responsible for converting chorismate to isochorismate and is necessary for the biosynthesis of both the respiratory chain component menaquinone (MK) and the Bacillus subtilis siderophore 2,3-dihydroxybenzoate (DHB) (Fig. 1). Although it is known that the carbon source, growth phase, and oxygen tension have regulatory effects, the actual signals that induce expression of respiratory chain components are unknown (37). However, the production of DHB is known to be regulated by iron concentration (1,25,26). Therefore, isochorismate is required by cells under very different environmental conditions.B. subtilis has two distinct isochorismate synthases encoded by the menF (30) and dhbC (29) genes. MenF and DhbC are 47% identical at the DNA level and have 35% amino acid identity (29). menF is a promoter-proximal gene of the MK biosynthetic gene cluster located at 273Њ on the chromosome (30,38). MK is a lipophilic, nonprotein redox component mediating electron transfer between dehydrogenases and cytochromes (37). dhbC is the second gene of the DHB biosynthetic gene cluster located at 291Њ on the chromosome (29). Under conditions of iron deprivation, B. subtilis synthesizes DHB, a component of the specific transport system for the uptake of extracellular iron (1,25,26). This is not the only instance of gene duplication in B. subtilis. There are two thymidylate synthetases, TSaseA and TSaseB, that are encoded by the unlinked thyA and thyB genes, respectively (22). Both enzymes are functional in bacteria grown at 37ЊC or lower temperatures, but only TSaseA is active at 46ЊC (22). There are two different a-type terminal oxidases in B. subtilis that are encoded by separate loci (32). One of the oxidases is associated with a heme C-containing unit (32). The two membrane alkaline phosphatases of B. subtilis have substantial differences with respect to molecular weight, substrate sp...

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A variety of fluorescein di-beta-D-galactopyranoside (FDG)-based substrates were evaluated for measuring beta-galactosidase expression in bacteria. One substrate, 5-acetylamino-FDG (C2FDG), performed well in all bacteria tested, including the slow growing mycobacterium, Mycobacterium bovis BCG. The sensitivity of C2FDG in intact, viable BCG was similar to that of o-nitrophenyl-beta-D-galactopyranoside in cell lysates when used to measure lacZ reporter gene activity. C2FDG was approximately 70-fold more sensitive than green fluorescent protein (GFP) in BCG when assayed in a fluorescence plate reader, and comparable to GFP when measured by flow cytometry. These assays provide an important new alternative for the rapid measurement of reporter gene expression in viable bacteria.