Dataset Information

Diribonuclease activity eliminates toxic diribonucleotide accumulation

ABSTRACT: RNA degradation is a central process in cells that is vital for transcriptional regulation. The degradation process is initiated by endoribonucleolytic fragmentation of long RNA polymers, followed by exoribonucleolytic cleavage to release mononucleotides from the fragment ends. In this process, the degradation of short oligonucleotides, especially diribonucleotides, into mononucleotides requires specific enzymatic activity, which in Gram-negative bacteria is provided by oligoribonuclease (Orn). Orn is unique in that it is the only essential exoribonuclease in Escherichia coli. Recent studies have shown that Orn cleaves only diribonucleotides under physiological conditions, suggesting that the accumulation of dinucleotides poisons cells. Yet, related organisms, such as Pseudomonas aeruginosa, display a growth defect but are viable without Orn, contesting its essentiality. Here, we took advantage of P. aeruginosa orn mutants to elucidate the mechanisms of their survival. Genetic screening for suppressors that restored colony morphology identified yciV. Purified YciV exhibited diribonuclease activity. YciV is present in all g-proteobacteria, suggesting that YciV from P. aeruginosa is distinct from its E. coli ortholog. Phylogenetic analysis revealed differences between the two orthologs that were mapped to the active site of the enzyme and correlated with differences in substrate profiles. The expression of P. aeruginosa YciV in E. coli eliminated the necessity of orn. Similarly, the deletion of yciV from P. aeruginosa caused orn to become essential. Together, these results show that diribonuclease activity is required in g-proteobacteria and that diribonucleotides are utilized to monitor the efficacy of RNA degradation. Because Orn is found in higher eukaryotes, these observations indicate a conserved mechanism for monitoring RNA degradation.

INSTRUMENT(S):

ORGANISM(S): Bacteria Pseudomonas Aeruginosa Pa14

TISSUE(S): Logarithmic Phase Culture

SUBMITTER: Conor Jenkins

LAB HEAD: Vincent T. Lee

PROVIDER: PXD052373 | Pride | 2024-09-16

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 21

Publications

Diribonuclease activity eliminates toxic diribonucleotide accumulation.

Kim Soo-Kyoung SK Orr Mona W MW Turdiev Husan H Jenkins Conor C CC Lormand Justin D JD Myers Tanner M TM Burnim Audrey Andy AA Carter Jared A JA Kung Warren C WC Jiang Xiaofang X Sondermann Holger H Winkler Wade C WC Lee Vincent T VT

Cell reports 20240913 9

RNA degradation is a central process required for transcriptional regulation. Eventually, this process degrades diribonucleotides into mononucleotides by specific diribonucleases. In Escherichia coli, oligoribonuclease (Orn) serves this function and is unique as the only essential exoribonuclease. Yet, related organisms, such as Pseudomonas aeruginosa, display a growth defect but are viable without Orn, contesting its essentiality. Here, we take advantage of P. aeruginosa orn mutants to screen f ...[more]

PMID: 39276351

Similar Datasets

Project description:Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives were unstable in microbial community due to the widespread of diverse oxygenases that could quickly degrade them. Hence, we sought to identify novel non-toxic, stable, and potent indole derivatives from plant sources for inhibiting biofilm formation of E. coli O157:H7 and P. aeruginosa PAO1. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN inhibited biofilms more effectively than indole for both E. coli and P. aeruginosa. Additionally, IAN decreased the production of virulence factor pyocyanin in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, while IAN induced indole-related genes and prophage genes in E. coli. It appears that IAN inhibits biofilm formation of E. coli by reducing curli formation and inducing indole production. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa. For the microarray experiments, P. aeruginosa were inoculated in 25 ml of LB medium in 250 ml shake flasks with overnight cultures that were diluted 1:100. IAN (100 μg/ml) dissolved in 25 μl DMSO or 25 μl DMSO alone as a control was added. Cells were cultured in LB at 37C with 250 rpm shakingfor 5 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

Project description:RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (two-fold, p<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains. In this study, we characterized the RpoS regulon of E. coli O157:H7 strain EDL933 using microarray analysis. A precise rpoS deletion mutant of EDL933 was constructed and employed in this study. EDL933 wild type and rpoS mutants were inoculated in triplicate into LB media at a starting OD of 0.0001 and grown aerobically at 37C. Cultures were harvested at OD600 = 0.3 in exponential phase and OD600=1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Genome 2.0 Array according to Affymetrix's standard protocols.

			Action	DRS
	Comp_OEC_1-011.raw	Raw
	Comp_OEC_2-009.raw	Raw
	Comp_OEC_3-003.raw	Raw
	Comp_OPA_1-004.raw	Raw
	Comp_OPA_2.raw	Raw