# Spx inhibits expression of the SwrA•DegU master flagellar activator in Bacillus subtilis

## TL;DR

The study reveals that Spx limits flagellar gene expression in Bacillus subtilis by repressing key activators, and ClpX protease helps control this by degrading Spx.

## Contribution

The novel finding is that Spx represses flagellar gene transcription, and ClpX protease regulates this repression by degrading Spx.

## Key findings

- Spx represses transcription of degU and swrA genes, which are key for flagellar biosynthesis.
- ClpX protease degrades Spx, relieving its repression and promoting swarming motility.
- Spx limits DegU-mediated positive feedback to prevent excessive flagellar biosynthesis under stress.

## Abstract

Flagella are trans-envelope nanomachines expressed from genes organized in a complex regulatory hierarchy governed at the highest level by transcription factors called master activators. The master activator of flagellar biosynthesis in Bacillus subtilis is a hybrid of SwrA•DegU that is required to increase flagellar density to swarm over solid surfaces. Here, we show that the ClpX unfoldase subunit of the ClpP protease is required for swarming motility, and that cells mutated for ClpX fail to swarm due to restricted levels of both SwrA and DegU. Suppressor mutations were found that increased expression of the fla/che operon under SwrA•DegU control, and mutation of the LonA protease elevates the levels of SwrA protein, while mutation of the global transcriptional regulator Spx increases transcription of both the degU and swrA genes. We conclude that ClpX promotes swarming motility via degradation of Spx, which represses motility gene transcription, including the Pfla/che, PdegU, and PswrA promoters, each activated by DegU. The ClpX-dependent regulatory proteolysis of Spx is relieved under stress conditions, and we infer that Spx may dampen DegU-mediated positive feedback to limit cell envelope stress caused by excessive flagellar biosynthesis.

Bacterial flagella are elaborate machines that are inserted through the bacterial cell envelope. The regulation of flagellar gene expression is organized in hierarchical tiers that largely correlate with the order of flagellar assembly. Here, we show that the abundance of the master activator of flagellar gene expression in Bacillus subtilis is restricted when the transcription factor Spx accumulates. Spx is an unusual transcription factor, as it binds to RNA polymerase and renders it insensitive to transcriptional activators. We show that Spx interferes with flagellar promoters activated by the response regulator DegU and may do so to limit a DegU-mediated positive feedback loop.