Physiological Roles of an -specific σ Factor

The Gram-negative pathogen is considered an "urgent threat" to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. species contain the poorly characterized ECF "SigAb;" however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to species and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists of itself, the stringent response mediator, , and the uncharacterized small RNA, "." However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of or resulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in the operon, "" and "", have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that , , and are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of .