Roles of the DEAD-box helicase CshA and the response regulator DegU in the regulation of temperature-dependent flagellar motility gene expression in Listeria monocytogenes

Abstract

Listeria monocytogenes is a food-borne pathogen that causes severe disease, particularly in immunocompromised individuals, infants, and the elderly. L. monocytogenes persists as a saprophyte across diverse environmental niches, including soil, decaying vegetation, and food processing facilities, yet transitions to an intracellular pathogen upon transmission to humans through contaminated food. Bacterial gene expression is rapidly remodeled in response to these distinct environments. Adaptation to shifting environments partially relies on flagellar motility (swimming motility), which facilitates chemotaxis, biofilm formation, and host cell invasion. Unlike most flagellated bacteria, which deploy a hierarchical sigma factor-dependent cascade to sequentially activate flagellar gene classes, L. monocytogenes coordinates motility through three core regulators (MogR, GmaR, and DegU) in a non-hierarchical, temperature-dependent manner. At 37°C, the master repressor MogR binds flagellar gene promoters to silence transcription. Below 34°C, the anti-repressor GmaR binds and sequesters MogR away from DNA, derepressing motility genes. Full activation of the fliN-gmaR operon, which encodes GmaR itself along with structural and assembly components of the flagellum, also requires the orphan response regulator DegU. Furthermore, a prior genetic screen identified the CshA DEAD-box RNA helicase as an accessory factor necessary for transcription of fliN-gmaR, although its contribution has remained unresolved. Both ΔcshA and ΔdegU strains show reduced gmaR transcript levels, a lack of flagellar motility, and the absence of flagellin protein production under permissive conditions (30°C). However, co-immunoprecipitation and mass spectrometry indicate no direct interaction between CshA and DegU at 30°C, suggesting their contributions to fliN-gmaR expression are mechanistically distinct. To define the independent contributions of CshA and DegU to flagellar gene expression, I performed RNA-seq on wild-type, ΔcshA, ΔdegU, and ΔmogRΔdegU strains grown at 30°C. Strand-specific sequencing revealed that in ΔcshA and ΔdegU mutant backgrounds, sense strand (coding strand) transcripts across the fliN-gmaR operon are markedly reduced, while a striking accumulation of antisense (noncoding) transcripts emerges at the 5' region of the operon, spanning fliN-fliP-fliQ, well upstream of the gmaR coding sequence itself. This antisense signal originates from anti0677 (anti-fliQ), a previously annotated noncoding RNA that has been characterized as part of a sense-antisense excludon at the fliN-gmaR locus, but whose regulation has not been incorporated into models of flagellar gene expression. CshA is a DEAD-box RNA helicase with documented RNA:RNA duplex unwinding activity in other bacterial systems (B. subtilis and related Firmicutes). My dissertation studies have led to a model in which CshA may overcome antisense-mediated interference at the fliN-gmaR operon by resolving sense-antisense duplexes formed between the nascent transcript and anti0677. Therefore, CshA functions post-transcriptionally to facilitate flagellar motility. To identify the molecular pathways through which CshA and DegU contribute to fliN-gmaR expression, I isolated spontaneous suppressor mutants of ΔcshA and ΔdegU that restored swimming motility on soft agar at 30°C and identified the mutations by whole-genome sequencing. All ΔdegU suppressors converge on the same core promoter mutation in fliN-gmaR, suggesting it enhances overall promoter strength sufficient to bypass the requirement for DegU's activation activity. The increased promoter activity may also help the operon overcome anti0677 antisense interference, complementing CshA's post-transcriptional role in duplex resolution. DegU therefore contributes at the level of transcription initiation. ΔcshA suppressors, by contrast, fell into broader functional categories (metabolism, motility machinery, stress signaling), suggesting CshA is involved in multiple aspects of L. monocytogenes physiology. Together, this work integrates the previously described fliN-gmaR sense-antisense excludon into the regulatory model for flagellar motility. The convergence of all ΔdegU suppressors on a single core promoter mutation that bypasses the requirement for DegU, combined with the dispersed functional distribution of ΔcshA suppressors across metabolism, motility machinery, and stress signaling, supports a model in which CshA contributes post-transcriptionally to fliN-gmaR operon expression while DegU enhances transcriptional output at the fliN-gmaR promoter. These findings provide a framework for understanding how transcriptional and post-transcriptional regulators may jointly coordinate flagellar motility in response to environmental conditions in L. monocytogenes.