Supplementary Material for: Contribution of Extracytoplasmic Function Sigma Factors to Transition Metal Homeostasis in Cupriavidus metallidurans Strain CH34

Cupriavidus metallidurans strain CH34 is a highly metal-resistant bacterium that contains 11 sigma factors of the extracytoplasmic function (ECF) protein family, which can be subgrouped into the ECF:FecI 1, ECF:FecI 2, ECF:RpoE and ‘(ECF)’ clusters. To analyze the contribution of these 11 sigma factors to metal resistance, upregulation of the respective genes was measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). As determined by RT-PCR, the ECF sigma factor genes were part of two- to tetra-cistronic operons, each containing genes for the sigma factor plus one or two antisigma factors. The three sigma factors RpoJ, RpoK and RpoI (ECF:FecI 1 cluster) were upregulated by Cu(II) and Ni(II), and under conditions of iron depletion. The other 8 ECF sigma factor genes were not induced by iron depletion. Strong upregulation of rpoJ and rpoK under iron depletion in a ΔrpoI mutant strain and close vicinity of rpoI to genes involved in iron siderophore metabolism marked RpoI as the primary ECF sigma factor for siderophore-mediated iron uptake. Genes for RpoO, RpoL and RpoM (ECF:FecI 2 cluster) were not upregulated by transition metal cations and influenced metal resistance only weakly. Concerning the two ‘(ECF)’ group proteins, rpoQ was strongly upregulated by Cu(II) and deletion of rpoR led to a small decrease in copper resistance. Of the three ECF:RpoE-encoding genes, rpoP was not transcribed under the conditions tested, cnrH was upregulated by Ni(II) and essential for nickel resistance as known before. RpoE was required for full metal resistance of C. metallidurans. None of these 11 sigma factors was essential for metal resistance mediated by the cobalt, zinc and cadmium resistance determinant czc, or for its expression. However, RpoI was essential for siderophore production in C. metallidurans, and, in addition to the known role of CnrH in nickel resistance, RpoE, RpoI, RpoJ, RpoK and maybe also RpoQ are required for the outstanding transition metal resistance of this bacterium.