Panel B: Features of a typical TAT signal sequence where x represents any amino acid (adapted from ). The arrowheads indicate signal peptidase cleavage sites. Based on these findings, we compared the ability of our panel of WT and tat mutant strains to grow in the presence of the β-lactam Selleckchem MK5108 antibiotic carbenicillin. This was accomplished PRT062607 concentration by spotting equivalent numbers of bacteria onto agar plates supplemented with the antibiotic. For comparison, bacteria were
also spotted onto agar plates without carbenicillin. These plates were incubated for 48-hr at 37°C to accommodate the slower growth rate of tat mutants. In contrast to WT M. catarrhalis O35E, which is resistant to carbenicillin, the tatA (Figure 5A), tatB (Figure 5B), and tatC (Figure 5C) mutants were sensitive to the antibiotic. The introduction of plasmids containing a WT copy of tatA (i.e. pRB.TatA, Figure 5A) and tatB (i.e. pRB.TatB, Figure 5B) did not restore the ability of the tatA and tatB mutants to grow in the presence of carbenicillin, respectively. Resistance to the β-lactam was observed only when the tatA and tatB mutants were complemented with the plasmid specifying the entire tatABC locus (see pRB.TAT in Figure 5A and B), which is consistent with the results of the growth experiments presented in Figure 3. Introduction of the plasmid encoding BTSA1 cost only the WT copy of tatC (i.e. pRB.TatC) in the strain O35E.TC was sufficient to restore the growth
of this tatC mutant on medium supplemented with carbenicillin (Figure 5C). Of note, the tatC mutant of strain O12E was tested in this manner and the results were consistent with those obtained with O35E.TC (data not shown). In order to provide an appropriate control for these experiments, an isogenic mutant strain of M. catarrhalis O35E was constructed in which the
bro-2 gene was disrupted with a kanR marker. The mutant, which was designated O35E.Bro, grew at the same rate as the parent strain O35E in liquid medium (Figure 3C). As expected, the bro-2 mutant did not grow on agar plates containing carbenicillin (Figure 5C). Figure 5 Growth of the M. catarrhalis WT isolate O35E and tat mutant strains in the presence of the β-lactam antibiotic carbenicillin. The ability of tat mutants to grow in the presence PAK6 of carbenicillin (cab) was tested by spotting equivalent numbers of bacteria onto Todd-Hewitt agar plates supplemented with the antibiotic (TH + cab). As control, bacteria were also spotted onto agar plates without carbenicillin (TH). These plates were incubated for 48 hrs at 37°C to accommodate the slower growth rate of the tat mutants. Panel A: Growth of O35E is compared to that of its tatA isogenic mutant strain, O35E.TA, carrying the plasmid pWW115 (control), pRB.TatA (specifies a WT copy of tatA), and pRB.TAT (harbors the entire tatABC locus). Panel B: Growth of O35E is compared to that of its tatB isogenic mutant strain, O35E.