Global transcriptional response of Caulobacter crescentus to iron availability

Abstract Background In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. Results In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. Conclusions Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms.

We are grateful to Michael T. Laub for making the C. crescentus DNA microarray slides available and Carla Rosenberg lab for assistance with the microarray scanning. This work was supported by grant 470663/2011-1 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). During the course of this work, JFSN and RFL were supported by postdoctoral fellowships, grants 2007/56306-0 and 2008/52874-6, from São Paulo Research Foundation (FAPESP). MVM is partly supported by CNPq.

We are grateful to Michael T. Laub for making the C. crescentus DNA microarray slides available and Carla Rosenberg lab for assistance with the microarray scanning. This work was supported by grant 470663/20111 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). During the course of this work, JFSN and RFL were supported by postdoctoral fellowships, grants 2007/563060 and 2008/528746, from São Paulo Research Foundation (FAPESP). MVM is partly supported by CNPq.