Ectopic

expression of RecU in 8325-4recUi strain, through the addition of IPTG, resulted in the disappearance of the aberrant phenotypes (B). Scale bars 1 μm. Panel (C) shows a comparison of the phenotypes of control strain BCBHV008; 8325-4recU inducible mutant, incubated in the presence or absence of IPTG and 8325-4ΔrecU mutant. The presence of anucleate cells can be associated with chromosome segregation defects that result in one sister cell with two chromosomes and another with none. However, they could also arise as a result of DNA degradation caused by DNA guillotining by the septum or due to decreased DNA damage repair. We therefore tested the susceptibility of recU mutants to UV light and mitomycin C, both of which cause DNA lesions [32, 33]. Depletion of recU in the strain 8325-4recUi resulted selleck screening library in a 2-fold PF477736 nmr decrease in mitomycin C MIC (from 0.8 to 0.4 ng/ml), compared to the same strain grown in the presence of IPTG or to the control strain BCBHV008. Importantly, addition of IPTG recovered the MIC to wild-type levels. Similar results were obtained for the null mutant

strain 8325-4ΔrecU which had a 6-fold decrease in the mitomycin MIC compared to the parental strain. RecU depletion also caused S. aureus to become more sensitive to UV damage, since 10 sec of exposure time to UV light were sufficient to kill approximately 99% of the 8325-4recUi cells grown in the absence of ITPG but had no significant effect on BCBHV008 cells or 8325-4recUi cells grown in the presence of the inducer, which required 20 sec of UV exposures for similar decrease in cell viability (Figure  3). Taken together, these results indicate that RecU is required for DNA damage

repair in S. aureus and that its ectopic expression from the spa locus was sufficient to fully recover UV and mitomycin C resistance to wild type levels. Figure 3 RecU depletion in 8325-4 recU i strain leads to increased susceptibility to UV damage. Cultures of control strain BCBHV008 and recU inducible mutant 8325-4recUi showing serial dilutions from 10-2 (left) to 10-5 (right). 10 μl spots were placed on TSA agar, containing or not IPTG, and irradiated with a UV dose of 4 J/m2/sec for 0, 10, 20, 30 and 60 seconds. Plates were then incubated overnight and the number of CFU’s was counted. Absence of RecU leads 3-mercaptopyruvate sulfurtransferase to increased recruitment of the SpoIIIE DNA pump to the division septum SpoIIIE is a DNA pump crucial for moving DNA into the forespore of B. subtilis during sporulation [34]. During vegetative growth of B. subtilis this protein plays an important backup role when the chromosome fails to segregate prior to septum formation [35–37]. The presence of SpoIIIE foci localized near the center of the septum in a small fraction (~6%) of vegetatively growing B. subtilis cells is thought to reflect its role in Bafilomycin A1 chemical structure post-septational chromosome partioning [38].