Biology subjects

Kirkpatrick, C. L.

Publications and source records attributed to Kirkpatrick, C. L..

4 recordsLinked to original sources

bioRxiv2025

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

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.

microbiology↗

bioRxiv2025

Probing the zooarchaeological record across time and space for ancient pathogens

Zoonoses are among the greatest threats to human health, with many zoonotic pathogens believed to have emerged during prehistory. Palaeomicrobiological investigations of the zooarchaeological record hold potential to uncover the reservoirs, host ranges, and host adaptations of zoonotic pathogens but face challenges in identifying promising specimens and pathogen DNA preservation. We performed palaeopathological and genetic examinations of 346 skeletal elements from domesticated and wild animals collected from 34 Eurasian sites dating across the last six millennia. We identified 68 signatures of ancient (opportunistic) pathogens, including the important zoonotic pathogen Salmonella enterica, and found support that palaeopathological lesions provide guidance for specimen selection. For two pathogen species, Erysipelothrix rhusiopathiae and Streptococcus lutetiensis, we confirmed their ancient authenticity using phylogenetics, showcasing an approach to explore the relationship between ancient low-coverage genomes and their modern-day relatives. Our work presents a pathway to understanding prehistoric zoonotic diseases by integrating zooarchaeological, palaeopathological, and genetic data.

microbiology↗

bioRxiv2020

A toxin-antitoxin system associated transcription factor of Caulobacter crescentus can influence cell cycle-regulated gene expression during the SOS response

Toxin-antitoxin (TA) systems are widespread in bacterial chromosomes but their functions remain enigmatic. Although many are transcriptionally upregulated by stress conditions, it is unclear what role they play in cellular responses to stress and to what extent the role of a given TA system homologue varies between different bacterial species. In this work we investigate the role of the DNA damage-inducible TA system HigBA of Caulobacter crescentus in the SOS response and discover that in addition to the toxin HigB affecting cell cycle gene expression through inhibition of the master regulator CtrA, HigBA possesses a transcription factor third component, HigC, which both auto-regulates the TA system and acts independently of it. Through HigC, the system exerts downstream effects on antibiotic (ciprofloxacin) resistance and cell cycle gene expression. HigB and HigC had inverse effects on cell cycle gene regulation, with HigB reducing and HigC increasing the expression of CtrA-dependent promoters. Neither HigBA nor HigC had any effect on formation of persister cells in response to ciprofloxacin. Rather, their role in the SOS response appears to be as transcriptional and post-transcriptional regulators of cell cycle-dependent gene expression, transmitting the status of the SOS response as a regulatory input into the cell cycle control network via CtrA. ImportanceAlmost all bacteria respond to DNA damage by upregulating a set of genes that helps them to repair and recover from the damage, known as the SOS response. The set of genes induced during the SOS response varies between species, but frequently includes toxin-antitoxin systems. However, it is unknown what the consequence of inducing these systems is, and whether they provide any benefit to the cells. We show here that the DNA damage-induced TA system HigBA of the asymmetrically dividing bacterium Caulobacter crescentus affects the cell cycle regulation of this bacterium. HigBA also has a transcription factor encoded immediately downstream of it, named here HigC, which controls expression of the TA system and potentially other genes as well. Therefore, this work identifies a new role for TA systems in the DNA damage response, distinct from non-specific stress tolerance mechanisms which had been proposed previously.

microbiology↗

bioRxiv2019

Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

Rod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive {Delta}tipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in {Delta}tipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.\n\nAuthor summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

microbiology↗