Canonical and non-canonical EcfG sigma factors control the general stress response in Rhizobium etli

A core component of the alpha-proteobacterial general stress response (GSR) is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed alpha-proteobacterial genome sequences contain two or more copies of genes encoding sigma(EcfG) -like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti-sigma factor (NepR) and two-component response regulator (PhyR). So far, the widespread occurrence of additional, non-canonical sigma(EcfG) copies has not satisfactorily been explained. This study explores the hierarchical relation between Rhizobium etli sigma(EcfG1) and sigma(EcfG2), canonical and non-canonical sigma(EcfG) proteins, respectively. Contrary to reports in other species, we find that sigma(EcfG1) and sigma(EcfG2) act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. sigma(EcfG1) drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of sigma(EcfG2), accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel sigma(EcfG) targets. We propose a modified model for GSR regulation in R. etli, in which sigma(EcfG1) and sigma(EcfG2) function largely independently. Based on a phylogenetic analysis and considering the prevalence of alpha-proteobacterial genomes with multiple sigma(EcfG) copies, this model may also be applicable to numerous other species.