The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.

The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.

Arginase Is Inoperative in Developing Soybean Embryos1

Arginase (EC 3.5.3.1) transcript level and activity were measured in soybean (Glycine max L.) embryos from the reserve deposition stage to postgermination. Using a cDNA probe for a small soybean arginase gene family, no transcript was detected in developing embryos. However, arginase transcripts increased sharply on germination, reaching a maximum at 3 to 5 d after germination. There was low but measurable in vitro arginase specific activity in developing embryos (less than 6% of seedling maximum). During germination arginase specific activity increased in parallel with the sharply increasing arginase transcript level. Seedling arginase activity was largely localized in cotyledons. Arginase activity was assayed in vivo by measuring urea accumulation in a urease-deficient mutant. No urea was detected in developing embryos, whereas accumulated urea paralleled arginase specific activity and transcript level in germinating seedlings. As in planta embryos, cultured cotyledons did not accumulate urea when arginine (Arg) was provided with other amino acids in a “mock” seed-coat exudate. Arg as the sole nitrogen source was converted to urea but did not support cotyledon growth. There appeared to be a lack of recruitment of the low-level arginase activity to hydrolyze free Arg in developing embryos, thus avoiding a futile urea cycle.

The GroES homolog of Helicobacter pylori confers protective immunity against mucosal infection in mice.

Helicobacter pylori is an important etiologic agent of gastroduodenal disease. In common with other organisms, H. pylori bacteria express heat shock proteins that share homologies with the GroES-GroEL class of proteins from Escherichia coli. We have assessed the heat shock proteins of H. pylori as potential protective antigens in a murine model of gastric Helicobacter infection. Orogastric immunization of mice with recombinant H. pylori GroES- and GroEL-like proteins protected 80% (n = 20) and 70% (n = 10) of animals, respectively, from a challenge dose of 10(4) Helicobacter felis bacteria (compared to control mice, P = 0.0042 and P = 0.0904, respectively). All mice (n = 19) that were immunized with a dual antigen preparation, consisting of H. pylori GroES-like protein and the B subunit of H. pylori urease, were protected against infection. This represented a level of protection equivalent to that provided by a sonicated Helicobacter extract (P = 0.955). Antibodies directed against the recombinant H. pylori antigens were predominantly of the IgG1 class, suggesting that a type 2 T-helper cell response was involved in protection. This work reports a protein belonging to the GroES class of heat shock proteins that was shown to induce protective immunity. In conclusion, GroES-like and urease B-subunit proteins have been identified as potential components of a future H. pylori subunit vaccine.