Physiological roles of an -specific σ factor.

UNLABELLED

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-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, "SabS." 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. Furthermore, 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 .

IMPORTANCE

is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the -specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.