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bioRxiv · 10.64898/2025.12.09.693126

An ancestral transmembrane transcription factor couples cell envelope regulation and the SOS response in Caulobacter crescentus

Brejndal, K. A.·Hansen, N. V.·Ghosh, K.·Nielsen, S.·Hansen, L. H.·Jakobsen, L. A.·Larsen, M. R.·Kirkpatrick, C. L.
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Abstract

The DNA damage (SOS) response in bacteria involves derepression of a set of genes in order to activate mechanisms to tolerate stress, repair DNA and slow down cell division. While some of these genes are well characterized, many genes exist which are clearly induced by DNA damage but for which the function is unclear. In Caulobacter crescentus, the toxin-antitoxin (TA) system higBA and a closely associated downstream transcription factor (higX) are strongly induced as part of the SOS response, but the role of higX is unknown. We show that, unexpectedly, HigX functions independently of HigBA as a cell membrane-associated regulator and is toxic when overexpressed in filamentous cells. ChIP-Seq indicated that it binds to several promoters associated with cell envelope regulation. In cells with the SOS response constitutively activated, HigX was overproduced, but at the same time was unable to bind the majority of its target promoters. higX was highly conserved in genomic context among many alpha-proteobacteria, while higBA was only found upstream of it in a small number of Caulobacter genomes, including the universal laboratory strain C. crescentus NA1000. Compositional analysis suggested that higBA originated from a foreign source, while higX is likely ancestral to the alpha-proteobacteria. Our data support a model where dysregulation of HigX production and activity in filamentous {Delta}lexA cells contributes to cell envelope instability and antibiotic sensitivity. Thus, the protective effect of inhibiting cell division during the SOS response in order to repair the DNA, carries the hidden cost of interference with HigX-mediated cell envelope maintenance. Author summaryThe DNA damage (SOS) response in bacteria is key for their survival in harsh conditions, but the genes which are induced by this response and the molecular mechanisms involved are variable between different bacteria. We investigated the function of an uncharacterized SOS-induced gene in Caulobacter crescentus (higX) and found that it is a membrane-localized DNA binding protein, but that it did not help survival in DNA damage. Instead, it negatively affected cell viability when induced, unlike other SOS response genes. Genomic analysis showed that higX is highly conserved among alphaproteobacteria, but it is only under SOS control in a small number of genomes (including Caulobacter crescentus) where a toxin-antitoxin system and its SOS-regulatory promoter have been inserted upstream. This work uncovers HigX as a novel factor in cell envelope regulation, and shows that loss of cell shape regulation, eg. in filamentous {Delta}lexA cells, can influence the ability of transmembrane transcription factors to bind to their target genes.

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Brejndal, K. A., Hansen, N. V., Ghosh, K., Nielsen, S., Hansen, L. H., Jakobsen, L. A., Larsen, M. R., Kirkpatrick, C. L.. 2025-12-09. An ancestral transmembrane transcription factor couples cell envelope regulation and the SOS response in Caulobacter crescentus. https://doi.org/10.64898/2025.12.09.693126

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