skip to main content

Explore Research Products in the NSF-PAR

Title: Sulfur Availability Impacts Accumulation of the 2-Thiouridine tRNA Modification in Bacillus subtilis
ABSTRACT Posttranscriptional modifications to tRNA are critical elements for the folding and functionality of these adaptor molecules. Sulfur modifications in tRNA are installed by specialized enzymes that act on cognate tRNA substrates at specific locations. Most studied organisms contain a general cysteine desulfurase to mobilize sulfur for the synthesis of S-tRNA and other thio-cofactors. Bacillus subtilis and other Gram-positive bacteria encode multiple cysteine desulfurases that partner with specific sulfur acceptors in the biosynthesis of thio-cofactors. This metabolic layout suggests an alternate mode of regulation in these biosynthetic pathways. In this study, tRNA modifications were exploited as a readout for the functionality of pathways involving cysteine desulfurases. These analyses showed that the relative abundance of 2-thiouridine-modified tRNA (s 2 U) responds to sulfur availability in the growth medium in a dose-dependent manner. This study found that low sulfur concentrations lead to decreased levels of the s 2 U cysteine desulfurase YrvO and thiouridylase MnmA, without altering the levels of other cysteine desulfurases, SufS, NifS, and NifZ. Analysis of pathway metabolites that depend on the activity of cysteine desulfurases indicates that sulfur nutrient availability specifically impacts s 2 U accumulation while having no effect on the levels of other S-modified tRNA or activity levels of Fe-S enzymes. Collectively, these results support a model in which s 2 U tRNA serves as a marker for sulfur availability in B. subtilis . IMPORTANCE The 2-thiouridine (s 2 U) tRNA modification is found ubiquitously across all domains of life. YrvO and MnmA, the enzymes involved in this modification, are essential in B. subtilis , confirming the well-established role of s 2 U in maintaining translational efficiency and, consequently, cellular viability. Herein, we show that in the model Gram-positive organism Bacillus subtilis , the levels of s 2 U are responsive to sulfur availability. Downregulation of the s 2 U biosynthetic components leads to lower s 2 U levels, which may serve as a signal for the slowing of the translational apparatus during cellular nutrient insufficiency. Our findings provide the basis for the identification of a potential bacterial mode of regulation during S-metabolite depletion that may use s 2 U as a marker of suboptimal metabolic status.  more » « less
Award ID(s):
1716535
NSF-PAR ID:
10344643
Author(s) / Creator(s):
; ;
Editor(s):
Shank, Elizabeth Anne
Date Published:
Journal Name:
Journal of Bacteriology
Volume:
204
Issue:
5
ISSN:
0021-9193
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ( , Methods in molecular biology)
    Dos Santos, P.C. (Ed.)
    Iron-Sulfur (Fe-S) clusters function as core prosthetic groups known to modulate the activity of metalloenzymes, act as trafficking vehicles for biological iron and sulfur, and participate in several intersecting metabolic pathways. The formation of these clusters is initiated by a class of enzymes called cysteine desulfurases, whose primary function is to shuttle sulfur from the amino acid l-cysteine to a variety of sulfur transfer proteins involved in Fe-S cluster synthesis as well as in the synthesis of other thiocofactors. Thus, sulfur and Fe-S cluster metabolism are connected through shared enzyme intermediates, and defects in their associated pathways cause a myriad of pleiotropic phenotypes, which are difficult to dissect. Post-transcriptionally modified transfer RNA (tRNA) represents a large class of analytes whose synthesis often requires the coordinated participation of sulfur transfer and Fe-S enzymes. Therefore, these molecules can be used as biologically relevant readouts for cellular Fe and S status. Methods employing LC-MS technology provide a valuable experimental tool to determine the relative levels of tRNA modification in biological samples and, consequently, to assess genetic, nutritional, and environmental factors modulating reactions dependent on Fe-S clusters. Herein, we describe a robust method for extracting RNA and analytically evaluating the degree of Fe-S-dependent and -independent tRNA modifications via an LC-MS platform. 
    more » « less
  2. ; ; ; ( , RSC Chemical Biology)
    null (Ed.)
    Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety of sulfur-modified nucleosides and nucleotides. While the discovery, regulation and functions of DNA phosphorothioate (PS) modification, where one of the non-bridging oxygen atoms is replaced by sulfur on the DNA backbone, are important topics, this review focuses on the sulfur modification in natural cellular RNAs and therapeutic nucleic acids. The sulfur modifications on RNAs exhibit diversity in terms of modification locations and cellular functions, but the various sulfur modifications share common biosynthetic strategies across RNA species, cell types and all domains of life. The first section reviews the post-transcriptional sulfur modifications on nucleobase with emphasis on thiouridine on tRNA and phosphorothioate modification on RNA backbones, as well as the functions of the sulfur modifications on different species of cellular RNAs. The second section reviews the biosynthesis of different types of sulfur modifications and summarizes the general strategy for the biosynthesis of sulfur-containing RNA residues. One of the main goals of investigating the sulfur modifications is to enrich the genomic drug development pipeline and enhance our understandings of the rapidly growing nucleic acid-based gene therapy. The last section of the review focuses on the current drug development strategies employing sulfur substitution of oxygen atoms in therapeutic RNAs. 
    more » « less
  3. ; ; ; ; ( , Nature Communications)
    Abstract

    The Gram-positive bacteriumBacillus subtilisexhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest intoB. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should makeB. subtilis easier to engineer in the future.

     
    more » « less
  4. ; ; ; ; ; ; ; ( , Journal of Bacteriology)
    ABSTRACT Biofilm development in Bacillus subtilis is regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate of sinR , which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, how B. subtilis regulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development in B. subtilis . IMPORTANCE In Bacillus subtilis , biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing. 
    more » « less
  5. ; ; ; ; ; ; ( , ChemBioChem)
    Abstract

    Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2‐thiouridine using selenophosphate or geranyl‐pyrophosphate as cofactors. In this study, we explored thein vitrogeranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U35was mutated to A35(ASL‐tRNALys(s2U)UUto ASL‐tRNAIle(s2U)AU). By examining the SelU cofactor geranyl‐pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl‐ and farnesyl‐pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end‐point analysis for SelU follows the order of ASLLys(s2UUU)ASLGln(s2UUG)>ASLGlu(s2UUC). These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU‐based antibiotics.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email