An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli

GENERAL INFORMATION:

This is the dataset that supports the findings of the manuscript "An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli ", currently published on eLife (Reviewed Preprint v1 May 15, 2024)

The dataset contains three main data types:

- Fluorescence_microscopy_data: Data obtained from single-molecule fluorescent in situ hybridisation (FISH) and Halo-labelling (HaloTag) experiments: raw images (.TIF files), examples of masks obtained from cell segmentation (.MAT files) and numerical data for quantification of foci in Escherichia coli cells (.CSV files).  

- Fluorescence_microscopy_SRC: source code used for image analysis and spot detection in FISH and HaloTag experiments (.MAT files) 

- Growth-and-viability_data: Data from cell growth and viability assays obtained from optical density measurements at 600 nm (OD600) and survival factor calculated using colony forming units per ml  (.CSV files) 

- RT-qPCR_data: Data from reverse transcription quantitative real-time PCR  (.CSV files)

Within data types, the data is divided in a "folder by figure" structure, meaning the data is separated according to the order that they appear in the publication figures (the numbers in the folder or file titles correspond to the figure numbers in the publication).  

Specific README files for each data type containing more detailed information can be found here and within each main data type folder. See README-FM, README-GV, README-PCR and README-SRC for fluorescence microscopy data, growth and viability data, RT-qPCR data and analysis code for microscopy images, respectively.

ABSTRACT:  All living organisms have developed strategies to respond to chromosomal damage and preserve genome integrity. One such response is the repair of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA lesions. In Escherichia coli, DSBs are repaired via RecBCD-dependent homologous recombination. RecBCD is essential for accurate chromosome maintenance, but its over-expression can lead to reduced DNA repair ability. This apparent paradox suggests that RecBCD copy numbers may need to be tightly controlled within an optimal range. Using single-molecule fluorescence microscopy, we have established that RecB is present in very low abundance at mRNA and protein levels. RecB transcription shows high fluctuations, yet cell-to-cell protein variability remains remarkably low. Here, we show that the post-transcriptional regulator Hfq binds to recB mRNA and down-regulates RecB protein translation in vivo. Furthermore, specific disruption of the Hfq-binding site leads to more efficient translation of recB mRNAs. In addition, we observe a less effective reduction of RecB protein fluctuations in the absence of Hfq. This fine-tuning Hfq-mediated mechanism might have the underlying physiological function of maintaining RecB protein levels within an optimal range.

LICENSES:

The data (microscopy, RT-qPCR and cell growth and viability assays) are shared under Creative Commons Attribution 4.0 International. 

The source code for spot detection and quantification analysis is under GNU-GPL license, and the source code for the bright-field segmentation pipeline is under MIT license.

For correspondence: irykalita@gmail.com, meriem.elkaroui@ed.ac.uk