Systems Level Analyses Reveal Multiple Regulatory Activities of CodY Controlling Metabolism, Motility and Virulence in Listeria monocytogenes

Abstract

Bacteria sense and respond to many environmental cues, rewiring their regulatory network to facilitate adaptation to new conditions/niches. Global transcription factors that co-regulate multiple pathways simultaneously are essential to this regulatory rewiring. CodY is one such global regulator, controlling expression of both metabolic and virulence genes in Gram-positive bacteria. Branch chained amino acids (BCAAs) serve as a ligand for CodY and modulate its activity. Classically, CodY was considered to function primarily as a repressor under rich growth conditions. However, our previous studies of the bacterial pathogen Listeria monocytogenes revealed that CodY is active also when the bacteria are starved for BCAAs. Under these conditions, CodY loses the ability to repress genes ( e. g., metabolic genes) and functions as a direct activator of the master virulence regulator gene, prfA. This observation raised the possibility that CodY possesses multiple functions that allow it to coordinate gene expression across a wide spectrum of metabolic growth conditions, and thus better adapt bacteria to the mammalian niche. To gain a deeper understanding of CodY’s regulatory repertoire and identify direct target genes, we performed a genome wide analysis of the CodY regulon and DNA binding under both rich and minimal growth conditions, using RNA-Seq and ChIP-Seq techniques. We demonstrate here that CodY is indeed active ( i. e., binds DNA) under both conditions, serving as a repressor and activator of different genes. Further, we identified new genes and pathways that are directly regulated by CodY (e.g., sigB, arg, his, actA, glpF, gadG, gdhA, poxB, glnR and fla genes), integrating metabolism, stress responses, motility and virulence in L. monocytogenes. This study establishes CodY as a multifaceted factor regulating L. monocytogenes physiology in a highly versatile manner.

Author Summary

Bacterial pathogens sense multiple host-related metabolic signals that alert them of host localization and result in induction of virulence traits. The Gram-positive foodborne pathogen Listeria monocytogenes activates the transcription of its virulence genes in response to low levels of branch-chained amino acids (BCAAs). This phenomenon is dependent on the global transcription regulator CodY, which binds BCAAs as a ligand and this binding affects its regulatory functions. CodY is classically thought to function under rich growth conditions, when bound to its ligands, however we recently reported that CodY directly activates L. monocytogenes virulence when BCAAs are limited. Identifying this novel CodY activity prompt us to further investigate CodY functions under different growth conditions in a genome wide level. For this purpose, we set on analyzing CodY’s regulon in L. monocytogenes in both rich and minimal growth conditions using genome-wide sequencing techniques. Remarkably, we identified for the first time a global regulatory role for CodY when BCAAs are limited, that are similar to those within the mammalian niche. Furthermore, our data establish CodY as a central regulator that integrates metabolism, motility, stress responses and virulence in L. monocytogenes.