Figure 4 Transcriptional fusion assays and the rhizobactin operon. (A) GusA activities were measured for fusions in genes rhtX, rhbB and rhbF in wild-type (Rm1021) and chvI261 mutant (SmUW38) strain backgrounds. (B) The rhizobactin genes are clustered

in one operon, F1 F2 and F3 represent the positions #Defactinib randurls[1|1|,|CHEM1|]# of the fusions to rhtX, rhtB, and rhbF respectively. The grey boxes (B1 and B2) represent the possible position for ChvI binding, and P1 and P2 are predicted promoters. The high basal level of the negatively regulated operons is not really unexpected given that we do not know the repressing conditions, and also the likelihood of multiple regulatory systems acting on these genes. These experiments involved the comparison of gene expression in genetic backgrounds that resulted in differences only in the presence / absence of the ChvI regulator. Otherwise, the environmental conditions

were not altered. Discussion An adaptation of methods to perform gel electrophoresis mobility shift assays allowed us to identify DNA fragments with higher affinity for ChvI. Analyses of these results force us to revise our earlier perceptions following phenotypic analyses of ExoS/ChvI as mainly a regulatory system for exopolysaccharide production. Our results suggest that the ChvI regulon includes genes from diverse pathways. Moreover, ChvI appears to have a dual regulatory role, activating and repressing different operons. The total number of targets likely far outnumbers the 27 fragments that we pulled out in our screen, especially considering that we did not hit the same fragment more than once, and we also did not MDV3100 mouse find a few other targets that had previously been shown to be bound by ChvI. The approach used in our study is highly complementary to the microarray and directed DNA binding study of Chen et al. [17] that resulted in the identification of several potential regulatory targets of ExoS/ChvI and the prediction of a consensus binding sequence. It is important to note, however, that of 19 upstream regions tested, binding was only detected

to three (ropB1, SMb21440, SMc01580), and a putative consensus sequence was determined using some upstream regions to which binding had not been demonstrated. Confirmation of this consensus binding sequence awaits more detailed DNA footprinting experiments on a larger number of identified targets. It is possible that Silibinin many ChvI-repressed genes may not have been detected in that study due to the use of a constitutively activated variant of the ChvI protein that might not have been able to function as a repressor. The binding of ChvI within SMa2337 (rhtX) to repress rhtXrhbABCDEF gene transcription could suggest that following the sensing of a signal other than the presence of iron, ExoS/ChvI represses genes for rhizobactin 1021 production. This operon is known to be upregulated by RhrA in iron-depleted conditions [31] and downregulated by RirA in iron-replete conditions [32].