Figure 6 Protein carbonylation levels in R1 (black bars) and mntE – (white bars). Cells (OD600 = 0.8) were harvested and treated with 40 mM H2O2 for 30 min. The protein carbonylation levels were determined by the DNPH assay. Data represent the means ± standard deviations of three independent experiments. Conclusions Although it is known that the Mn/Fe ratio of D. radiodurans is higher than that of other bacteria, little is known regarding the maintenance of the

intracellular manganese ion level in this bacterium. So far, only one manganese efflux this website system has been identified in bacteria [10], and it is still unknown Epigenetics inhibitor whether this system exists in D. radiodurans [22]. In this study, we identified a MntE homolog in D. radiodurans. As expected, our results showed that the intracellular

manganese ion level was almost four-fold higher in the mutant than in R1. Furthermore, we also found that the oxidative level of mntE – proteins decreased to almost one half that of R1. On the other hand, the data also revealed that manganese accumulation is dangerous to the mntE – mutant. Based on these data, we conclude that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The results provide additional evidence that intracellular manganese ions are involved in the radiation resistance learn more of D. radiodurans. However, because the intracellular Mn/Fe ratio and the Mn concentration of mntE – both increased in this study, we could not clarify whether the Mn/Fe ratio or the Mn concentration is more important for stress tolerance. Therefore, global analysis of the regulation of the intracellular manganese ion level is necessary in further studies. Methods Strains and media All the strains and plasmids used in this study are Amino acid listed in the supporting information (Table 1). The D. radiodurans strains were cultured at 30°C in TGY (0.5% Bacto tryptone, 0.1% glucose, and 0.3% Bacto yeast extract) medium with aeration

or on TGY plates supplemented with 1.2% Bacto agar. Table 1 Strains and plasmids used in this study Strain or plasmid Relevant marker Reference or resource Strains     E. coli DH5α hsdR17 recA1 endA1 lacZΔM15 Invitrogen D. radiodurans R1 ATCC13939   mntE – As R1, but mnE::aadA This study mntR As mntE – mnE::aadA(pME mntE Dr +) This study Plasmids     pMD18-T TA cloning vector Takara pRADK E. coli-D. radiodurans shuttle vector carrying D. radiodurans groEL promoter [27] pME pRADK derivative expressing D. radiodurans mntE This study Disruption and complementation of dr1236 The mutant dr1236 gene was constructed as described previously [23]. Briefly, ~600-bp DNA fragments immediately upstream and downstream from dr1236 were amplified from the genome of the R1 strain using the primer pairs ME1/ME2 and ME3/ME4, respectively (Table 2).