While UreI presents a total of fourteen protonable residues, Yut has only three, and UreT possesses seven (data not shown). The higher number of protonable residues of UreT could account for the differences found in acid activation between Yut and UreT. However, the mechanism of urea selectivity is probably the same, as a comparison with the crystal structure of the urea transporter of D. vulgaris shows that all the residues that form the pore are conserved (data not shown). The only one minor difference is that in one of the two urea slots present in UreT, one of the phenylalanines forming the slot is changed to leucine (L201F), and the corresponding
leucine in the slot is changed to phenylalanine (F304L) (data not shown). Since urea uptake is not pH regulated in Yersinia spp, the buy Vactosertib unrestricted
entry of urea would alkalinize the cytoplasm to lethal levels. Yersinia has solved this problem by expressing a urease with check details an acidic pH-optimum, that has little or no activity at ~pH 8.0 . Brucella urease has a pH optimum of 7.3, and although its activity is much lower at pH 8.0, it is still significant. In this case, the problem of lethal alkalinization is prevented by the existence of a pH-regulated urea transporter that reduces urea uptake to just the amount that diffuses through the inner membrane. In contrast to the ΔureT mutant, mutants ΔureTp and ΔnikO showed ZD1839 order around a 40% decrease in urease activity in cell extracts. Both phenotypes were reversed by complementation of the mutant strains with a nikO-containing plasmid or, alternatively, with high concentrations of nickel in the culture Cell press medium suggesting that the amount of active urease in these mutants was limited by nickel availability. Complementation of the urease activity of the ΔureTp mutant with the nikO plasmid was rather surprising if we
consider that the mutant should be defective not only in nikO but also in the other nik genes. Furthermore, the susceptibity to low pH of the ΔureTp mutant was not complemented by the nikO gene in trans, suggesting that other factors may be implicated in the acid resistance phenotype of Brucella. NikO is predicted to be the ATPase component of an ECF-type nickel transporter, and its mutation should abolish most of the activity of the transporter. There is another nickel transport system already described in B. suis, NikABCDE (10). nikA mutants were not affected in urease activity unless a chelating agent was added to the medium. As both the ΔureTp and ΔnikO mutants show lower urease activity than the wild type when grown in standard medium, we concluded that NikKMLQO is the main nickel transport system in Brucella. B. suis nikA mutants have an intact NikKMLQO nickel transporter, whose function can override the nikA mutation. In B. abortus 2308 by contrast, the single nikO mutation produced a significant decrease in urease activity. Sequence analysis reveals that the three B.