Mutants ΔureTp and ΔnikO were complemented by the nikO containing

Mutants ΔureTp and ΔnikO were complemented by the nikO containing plasmid pFJS245 (Figure 2A), but no effect on urease activity was observed when pFJS243 (containing ureT) was used

to complement the ΔureTp, ΔureT, or ΔnikO mutants (data not shown). Figure 2 Urease activity of B. abortus 2308-derived strains. Urease activity was determined in bacterial extracts obtained from the indicated strains and growth conditions, and expressed in μmol of NH3 min-1 mg-1 protein The experiments were performed by triplicate with three technical measures per replica. The data shown correspond to one representative experiment and the error bar indicates the standard deviation. An unpaired t-test was performed to determine if the urease activity of each Epigenetics inhibitor mutant was significantly different than the corresponding wild type control. A. Protein extracts from cultures of the indicated strains this website grown in BB. B: Protein extracts from cultures grown in BB supplemented with 0.5 mM NiCl2. * indicates p < 0.05. Effect of nickel addition on urease activity of different Brucella strains Nickel and cobalt are transition metals that can share the same bacterial import systems [16]. The genes nikKMLQO, currently annotated in the Brucella genomes as components of a cobalt transport system, are found downstream of the ure2 genes, and form part of the same operon, so we tested whether they were

involved in the transport of nickel, which is essential Janus kinase (JAK) for urease activity. The addition of an excess of nickel in the form of NiCl2 would supply the metal needed for urease assembly in spite of the Dorsomorphin molecular weight inactivation of the nik transport system. We tested the urease activity of the different strains grown in the presence or absence of 0.5 mM NiCl2 in the culture medium. The results

in Figure 2B indicate that the urease activity of all the mutants reverted to normal values when the culture medium was supplemented with nickel, thus confirming the suspected role of the products of the nik genes of the ure2 operon in nickel transport. These results are also a further evidence for the extension of the operon until the nikO gene; that is a polar ureT mutation has a lower urease activity than the corresponding non-polar mutant, and identical activity to that of the nikO mutant, suggesting that the observed phenotype is the result of a polar effect on the genes downstream of ureT. Effect of pH on urease activity in intact cells Brucella urease assayed in vitro shows a pH-dependent activity that is maximal at pH 7.3 [1]. When urease activity was assayed in intact B. abortus 2308 cells, the activity was higher at low pH values and dropped to near one third as the pH of the medium reached a value of 6 (Figure 3). ΔureT intact cells showed very similar activity to wild type cells at pH values above 6, but they lost the acid-dependent induction of urease activity at lower pH values.

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