The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), kef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pagklacZ transcriptional construct to measure pag promoter activity, we cloned in BaciUus subtilis a gene (alxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located aIxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcriptiontranslation analysis. The cloned airA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. 3-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.

Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by transduction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enterocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.

Two toxic polypeptides of 24 and 25 kilodaltons (kDa) were purified from parasporal proteinaceous crystals of Bacillus thuringiensis subsp. israelensis. Both of these polypeptides, which are antigenically similar and have identical N terminals, lysed human erythrocytes and cultured mosquito cells. Although the 24-kDa peptide was more toxic than the 25-kDa peptide, both were less toxic than the crude alkali-solubilized crystal toxin. However, a 1:1 mixture of these 24and 25-kDa proteins was more toxic than either of these polypeptides individually, indicating a possible interaction between these proteins at the cell membrane. Both the 24and the 25-kDa proteins were inactivated by aqueous suspensions of dioleolylphosphatidylcholine, indicating the involvement of phospholipids in the cytotoxic action of these toxins. Thus the role of cell membrane phospholipids in mediating the toxin action was studied by using phospholipases as probes. Treatment of erythrocytes with high levels of phospholipase D increased their susceptibility to the toxin; however, phospholipase A2-treated erythrocytes were less susceptible to the toxin. These erythrocytes also bound less 125I-labeled 25-kDa toxin. These results support the role of fatty acyl residues at the syn-2 position of membrane phospholipids in toxin action. The cytolytic toxin of B. thuringiensis subsp. israelensis is thought to damage cell membranes in a detergentlike manner. However, there was a difference between the cytolytic action of this toxin and that of a nonionic detergent such as Triton X-100 because phospholipase A2-treated erythrocytes were more susceptible to Triton X-100, whereas such erythrocytes were less sensitive to the toxin. Thus, the cytolytic toxin apparenty did not act as a nonspecific detergent, but rather interacted with phospholipid receptors on the cell membrane. Such an interaction of the toxin with phospholipid receptors probably results in the increased cell permeability, thereby causing cell lysis.

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