Table 2. Iron-sulfur cluster assembly genes induced by nitric oxide. Gene Ratio St.Dev. Protein Function Gene No. BMA2033 BMAA0021 BMAA0883 BMAA1800 BMA0666 BMA0667 BMA2729 BMA0610 BMA1706 BMA1707 BMA1708 BMA1709 BMAA1430 cysD-1 cysN sufS iscU iscS-1 iscR 14.1 2.7 4.6 4.5 3.5 4.3 2.2 3.8 3.3 2.3 3.9 6.9 2.0 1.4 1.3 2.7 3.0 0.7 0.5 0.4 1.7 0.1 0.7 0.5 2.9 0.5 iron compound ABC transporter siderophore-interacting protein iron permease, FTR1 family putative bacterioferritin-associated ferredoxin sulfate adenylyltransferase, subunit 2 sulfate adenylyltransferase, subunit 1 sulfur carrier protein cysteine desulfurase [Fe-S] assembly accessory protein [Fe-S] assembly scaffold cysteine desulfurase [Fe-S] assembly transcription factor [Fe-S] binding protein NO killing of B. mallei Pathway iron assimilation iron assimilation iron assimilation iron assimilation sulfur assimilation sulfur assimilation sulfur assimilation [Fe-S] assembly [Fe-S] assembly [Fe-S] assembly [Fe-S] assembly [Fe-S] assembly [Fe-S] assembly doi:10.1371/journal.pone.0001976.t002 activity of the [Fe-S] cluster protein aconitase was markedly reduced in B. mallei cultures treated with spermine NONOate. For instance, about 100 pmoles of NO per 106 bacteria completely inactivated the activity of aconitase (fig. 5A). To test more directly whether [Fe-S] clusters are a key target of the anti-B. mallei activity of RNS, an insertion mutant was constructed in the iscR transcriptional factor that regulates [Fe-S] cluster assembly. The lack of iscR increased the susceptibility of B. mallei to RNS generated by the NO donor spermine NONOate (fig. 5B). We next tested whether the iscR mutant is susceptible to NO produced by activated macrophages. Because wild-type B. mallei is already highly susceptible to the iNOS-mediated antibacterial activity of IFNc-primed macrophages after 5 h of infection (fig. 3B), the survival of wild-type and iscR-deficient B. mallei was compared after 2 h of challenge. As anticipated (fig. 5B), at this early time the iscR mutant was more susceptible than wild-type controls to the antimicrobial activity of IFNc-primed macrophages (fig. 5C). The difference in intracellular survival of wild-type and iscR-deficient bacteria was not as great in IFNc-primed macrophages lacking iNOS compared to those containing iNOS (fig. 5C). These data suggest that iscR mutant bacteria are hypersusceptible to the RNS generated by the host cells. The aconitase activity of the iscR mutant grown overnight in LBG medium was similar to that of wild-type controls (fig. 5D). However, the enzymatic activity of aconitase was more vulnerable to RNS-mediated inactivation in the absence of a functional iscR. Together, these findings are consistent with the notion that [Fe-S] clusters are key targets of the RNS-dependent bactericidal activity against B. mallei. Figure 5. iscR-deficient B. mallei is hypersusceptible to NO-mediated cytotoxicity. The aconitase activity of B. mallei exposed to spermine NONOate (NO) for 30 min can be seen in panel A. Panel B shows the cytotoxicity of 10 mM spermine NONOate against wild-type (WT) or iscR-deficient B. mallei. The intracellular survival of WT and isogenic iscR-deficient B. mallei was recorded in IFNc-treated macrophages isolated from wild-type C57BL/6 mice (B6) or iNOS-deficient congenic controls (C). The % survival after 2 h of infection was estimated according to the number of bacteria originally added to the macrophages. The aconitase activity present in overnight cultures of wild-type or iscR-deficient B. mallei is shown in D. Selected groups of bacteria were treated with 500 mM spermine NONOate for 30 min before the aconitase activity present in cytoplasmic extracts was recorded. Panels A & D represent data of two independent experiments. Data in panel B & C are the mean 6 sem from 4–8 independent observations. doi:10.1371/journal.pone.0001976.g005 PLoS ONE | www.plosone.org 6 April 2008 | Volume 3 | Issue 4 | e1976