Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes.

Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron. In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes. The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants [Grossman, T.H., Tuckman, M., Ellestad, S. and Osburne, M.S. (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B. subtilis sfpo and Escherichia coli entD genes. J. Bacteriol. 175, 6203-6211] were analyzed and five open reading frames were identified. These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs. Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29%. DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication. Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.

Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate.

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.

Direct cytotoxic action of Shiga toxin on human vascular endothelial cells.

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 effect 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 125I-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 125I-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.