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

2007-06-25T00:00:00Z (GMT) by Grosse C. Friedrich S. Nies D.H.
<i>Cupriavidus metallidurans</i> 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 <i>rpoJ</i> and <i>rpoK</i> under iron depletion in a Δ<i>rpoI</i> mutant strain and close vicinity of <i>rpoI</i> 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, <i>rpoQ</i> was strongly upregulated by Cu(II) and deletion of <i>rpoR</i> led to a small decrease in copper resistance. Of the three ECF:RpoE-encoding genes, <i>rpoP</i> was not transcribed under the conditions tested, <i>cnrH</i> was upregulated by Ni(II) and essential for nickel resistance as known before. RpoE was required for full metal resistance of <i>C. metallidurans</i>. None of these 11 sigma factors was essential for metal resistance mediated by the cobalt, zinc and cadmium resistance determinant <i>czc</i>, or for its expression. However, RpoI was essential for siderophore production in <i>C. metallidurans</i>, 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.