Ashby, E. | bioRxiv Science

bioRxiv2023

The timing of transcription of RpoS-dependent genes varies across multiple stresses

The alternative sigma factor RpoS regulates transcription of over 1000 genes in Escherichia coli in response to many different stresses. RpoS levels rise continuously after exposure to stress, and the consequences of changing levels of RpoS for the temporal patterns of expression of RpoS-regulated genes have not been described. We measured RpoS levels at various times during the entry to stationary phase, or in response to high osmolarity or low temperature, and found that the time required to reach maximum levels varied by several hours. We quantified the transcriptome across these stresses using RNA-seq. The number of differentially expressed genes differed among stresses, with 1379 DE genes identified in stationary phase, 633 in high osmolarity, and 302 in cold shock. To quantify the timing of gene expression, we fit sigmoid or double sigmoid models to differentially expressed genes in each stress. During the entry into stationary phase, genes whose expression rose earlier tended to be those that had been found to respond most strongly to low levels of RpoS. The timing of individual genes expression was not correlated across stresses. Taken together, our results demonstrate E. coli activates RpoS with different timing in response to different stresses, which in turn generates a unique pattern of timing of the transcription response to each stress. ImportanceBacteria adapt to changing environments by altering the transcription of their genes. Specific proteins can regulate these changes. This study explored how a single protein called RpoS controls how many genes change expression during adaptation to three stresses. We found that: 1) RpoS is responsible for activating different genes in different stresses; 2) that during a stress, the timing of gene activation depends on the what stress it is, and; 3) that how much RpoS a gene needs in order to be activated can predict when that gene will be activated during the stress of stationary phase.

microbiology↗

bioRxiv2022

Genomic occupancy of the bromodomain protein Bdf3 is dynamic during differentiation of African trypanosomes from bloodstream to procyclic forms

Trypanosoma brucei, the causative agent of Human and Animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism as it adapts to each host environment. These changes are reflected in the differing transcriptomes of parasites living in each host. While changes in the transcriptome have been well catalogued for parasites differentiating from the mammalian bloodstream to the insect stage, it remains unclear whether chromatin interacting proteins mediate transcriptomic changes during life cycle adaptation. We and others have shown that chromatin interacting bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stage parasites remains unknown. To address this question, we performed Cleavage Under Target and Release Using Nuclease (CUT&RUN) timecourse experiments using a tagged version of Bromodomain Protein 3 (Bdf3) in parasites differentiating from bloodstream to insect stage forms. We found that Bdf3 occupancy at most loci increased at 3 hours following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes known to have altered transcript levels during differentiation, such as procyclins, procyclin associated genes, and invariant surface glycoproteins. While most Bdf3 occupied sites are observed throughout differentiation, a very small number appear de novo as differentiation progresses. Notably, one such site lies proximal to the procyclin gene locus, which contains genes essential for remodeling surface proteins following transition to the insect stage. Overall, these studies indicate that occupancy of chromatin interacting proteins is dynamic during life cycle stage transitions, and provides the groundwork for future studies aimed at uncovering whether changes in bromodomain protein occupancy affect transcript levels of neighboring genes. Additionally, the optimization of CUT&RUN for use in Trypanosoma brucei may prove helpful for other researchers as an alternative to Chromatin Immunoprecipitation (ChIP).

molecular biology↗