The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Matsuo, Yoshitaka; Morimoto, Takahiro; Kuwano, Masayoshi; Loh, Pek Chin; Oshima, Taku; Ogasawara, Nagahisa

doi:10.1074/jbc.m512556200

Cited by 70 publications

(121 citation statements)

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“…Indeed, depletion of these proteins in bacterial cells leads to a decrease in 70S ribosomes with a buildup of 50S and 30S subunits (36,43,46). In contrast, both RbgA and HflX have been shown to bind to the 50S subunit and are required for its biogenesis, as cells depleted for RbgA show a reduction in 70S ribosomes, whereas free 50S subunits are completely missing (40,42,(47)(48)(49). HflX has also been implicated as a ribosome-splitting factor, involved in rescuing stalled ribosomes during stress (50).…”

Section: Resultsmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

187

227

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The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Grampositive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Grampositive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases-RsgA, RbgA, Era, HflX, and ObgE-as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.ribosome | stringent response | tolerance | ppGpp | Staphylococcus aureus

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Section: Resultsmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

187

227

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“…Although the basic mechanisms of late assembly of the 50S subunit differ between E. coli and B. subtilis, the 45S particles share some common properties. As in the E. coli 45S precursor, substoichiometric levels of L16, L27, and L36 are present in the 45S particle that accumulates in the B. subtilis rbgA mutant (36,37,41). In addition, cryo-EM structures revealed that H38 and 5S rRNA are not yet oriented and that domains IV and V are not properly built up, in the B. subtilis 45S particle (41,42).…”

Section: Discussionmentioning

confidence: 99%

“…RbgA/YlqF is an essential GTPase required for the proliferation of B. subtilis (36,37). Depletion of RbgA or expression of a dominant-negative mutant of RbgA resulted in accumulation of the 45S particles (36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Arai

Ishiguro

Kimura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.R ibosomes are essential ribonucleoprotein complexes that translate the genetic information encoded by mRNA into protein. Intact bacterial ribosomes, which have a sedimentation coefficient of 70S, consist of large (50S) and small (30S) subunits. Each subunit can be reconstituted in vitro from its individual component ribosomal RNAs and proteins (1-5), indicating that these components contain all of the information necessary to automatically assemble into functional subunits. Because the intermediate particles observed in an in vitro reconstitution of the subunits are similar to those observed in vivo, assembly maps that describe the order of ribosomal protein binding in vitro are useful tools for understanding ribosome biogenesis in the cell (6). However, assembly is slower in vitro than in vivo, and nonphysiological conditions, such as high-salt concentration and high temperature, are required for in vitro assembly.In the cell, ribosome assembly is coordinated with the transcription and processing of large precursor rRNAs encoded by the rrn operon (3, 7). Once transcription starts, nascent transcripts rapidly form local secondary and tertiary structures that serve as binding sites for the primary ribosomal proteins, thereby initiating ribosome assembly. However, hierarchical assembly starting at the 5′-terminal domains of the 16S and 23S rRNAs is not essential for ribosome formation in the cell (8) because nonribosomal factors, termed "assembly factors," facilitate rapid and efficient assembly of ribosomal particles. Ribosome biogenesis is an energy-consuming process in which a number of assembly factors, including RNA helicases, GTPases, protein chaperones, and rRNA-modifying enzymes, support efficient assembly of each subunit (6, 9, 10). Individual mutation or deletion of several assembly factors causes accumulation of immature 50S subunits that sediment at 40S or 45S. For example, the RNA helicase SrmB participates in the early stage of 50S assembly (11,12); deletion of srmB results in accumulation of a 40S ...

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“…It has been experimentally demonstrated that B. subtilis ylqF encodes a GTP-binding protein that participates in the late step of 50S ribosomal subunit assembly (Matsuo et al, 2006). Moreover, ylqF has been shown to be an essential gene in B. subtilis (Uicker et al, 2007).…”

Section: Results Lmo1273 Encodes An Rnase H Homologuementioning

confidence: 99%

“…The two distal genes of the locus (lmo1272 and lmo1273), which are co-transcribed with the SPase I genes (Raynaud & Charbit, 2005), encode proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, of Bacillus subtilis. In the latter organism, RnhB is involved in RNA-DNA heteroduplex degradation (Itaya et al, 1999;Ohtani et al, 1999a) and YlqF participates in ribosome biogenesis (Matsuo et al, 2006;Uicker et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

lmo1273, a novel gene involved in Listeria monocytogenes virulence

et al. 2009

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Listeria monocytogenes is a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome of L. monocytogenes EGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (lmo1272 and lmo1273), encoding proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, of Bacillus subtilis. lmo1273 could complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation of lmo1273 provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of the lmo1273 mutant in target organs was significantly impaired. However, the mutation did not impair L. monocytogenes intracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with an lmo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.

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The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Cited by 70 publications

References 50 publications

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

lmo1273, a novel gene involved in Listeria monocytogenes virulence

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