Riboswitch-Mediated Detection of Metabolite Fluctuations During Live Cell Imaging of Bacteria

Weiss, Cordelia A; Winkler, William E.

doi:10.1007/978-1-0716-1499-0_12

Cited by 3 publications

(1 citation statement)

References 57 publications

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“…It has been shown previously that an increase in GG leads to a concomitant increase in c-di-GMP through feedback inhibition. Previously, a double Δ nrnA Δ nrnB mutant was assayed for c-di-GMP levels using a fluorescent c-di-GMP riboswitch reporter ( 13 , 28 , 44 ). This revealed that levels of c-di-GMP, and consequently GG, were higher in this strain compared to wild-type.…”

Section: Resultsmentioning

confidence: 99%

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Weiss

Myers

et al. 2022

Nucleic Acids Research

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Bacterial RNases process RNAs until only short oligomers (2–5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2–4 nucleotides in length, all of which were processed from their 5′ terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

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Section: Resultsmentioning

confidence: 99%

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Weiss

Myers

et al. 2022

Nucleic Acids Research

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High content design of riboswitch biosensors: All-around rational module-by-module design

Zhu

et al. 2023

Biosensors and Bioelectronics

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Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways

Michaud,

Garneau,

Côté

et al. 2024

Sci Rep

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Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches.

show abstract

Riboswitch-Mediated Detection of Metabolite Fluctuations During Live Cell Imaging of Bacteria

Cited by 3 publications

References 57 publications

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

High content design of riboswitch biosensors: All-around rational module-by-module design

Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways

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