The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions

The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown.

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(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions

10.1101/2021.01.19.427108 ◽

2021 ◽

Author(s):

Daniel J. Bennison ◽

Jose A. Nakamoto ◽

Timothy D. Craggs ◽

Pohl Milón ◽

John B. Rafferty ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bound States ◽

Growth Control ◽

Stringent Response ◽

Ribosome Assembly ◽

Bacterial Cells ◽

Gtpase Activity ◽

70S Ribosome ◽

Ribosome Association

ABSTRACTDuring nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.

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Sleeping ribosomes: bacterial signaling triggers RaiA mediated persistence to aminoglycosides

10.1101/2020.11.27.401281 ◽

2020 ◽

Author(s):

Manon Lang ◽

Evelyne Krin ◽

Chloé Korlowski ◽

Odile Sismeiro ◽

Hugo Varet ◽

...

Keyword(s):

Sucrose Gradient ◽

Gradient Analysis ◽

Cell Formation ◽

Fold Increase ◽

Growth Conditions ◽

Lag Phase ◽

Bacterial Signaling ◽

Tryptophan Degradation ◽

70S Ribosome ◽

Persistent Cell

AbstractIndole is a small molecule derived from tryptophan degradation and proposed to be involved in bacterial signaling. We find that indole secretion is induced by sublethal tobramycin concentrations and increases persistence to aminoglycosides in V. cholerae. Indole transcriptomics showed strongly increased expression of raiA, a ribosome associated factor. Deletion of raiA abolishes the appearance of indole dependent persisters to aminoglycosides, while its overexpression leads to 100-fold increase of persisters, and a reduction in lag phase, evocative of increased active 70S ribosome content, which was confirmed by sucrose gradient analysis. We propose that, under stress conditions, inactive 70S ribosomes are associated with RaiA to be stored and rapidly reactivated when growth conditions become favorable again, in a mechanism different than ribosome hibernation. Our results point to an active process of persistent cell formation, through ribosome protection during translational stress and relief upon antibiotic removal. Translation is a universal process, and these results could help elucidate a mechanism of persistence formation in a controlled, thus inducible way.

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A Mutation of RNA Polymerase β′ Subunit (RpoC) Converts Heterogeneously Vancomycin-Intermediate Staphylococcus aureus (hVISA) into “Slow VISA”

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00135-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4215-4225 ◽

Cited By ~ 13

Author(s):

Miki Matsuo ◽

Tomomi Hishinuma ◽

Yuki Katayama ◽

Keiichi Hiramatsu

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Rna Polymerase ◽

Stringent Response ◽

Whole Genome ◽

Multicopy Plasmid ◽

Β Subunit ◽

Whole Genome Analysis ◽

Content Type ◽

Peptidoglycan Biosynthesis

ABSTRACTVarious mutations in therpoBgene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneously VISA (hVISA) strains. We reported thatrpoBmutations are also linked to the expression of the recently found “slow VISA” (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024–5035, 2014,http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of therpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, inrpoC. Introduction of the wild-type (WT)rpoCgene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that therpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: therpoCgene itself as regulatory mutations, peptidoglycan biosynthesis genes, andrelQ, which is involved in the stringent response. It appears that therpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

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Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Scientific Reports ◽

10.1038/s41598-020-73796-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Tanit Boonsiri ◽

Shinya Watanabe ◽

Xin-Ee Tan ◽

Kanate Thitiananpakorn ◽

Ryu Narimatsu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Quality Control ◽

Protein Quality ◽

Stringent Response ◽

Protein Quality Control ◽

Purine Biosynthesis ◽

Genome Comparison ◽

Whole Genome ◽

Intergenic Regions ◽

Mrsa Strains

Abstract Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

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Magnesium suppresses defects in the formation of 70S ribosomes as well as in sporulation caused by lack of several individual ribosomal proteins

10.1101/300103 ◽

2018 ◽

Author(s):

Genki Akanuma ◽

Kotaro Yamazaki ◽

Yuma Yagishi ◽

Yuka Iizuka ◽

Morio Ishizuka ◽

...

Keyword(s):

Growth Rate ◽

Cell Proliferation ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Living Cells ◽

Master Regulator ◽

Mutant Strains ◽

70S Ribosome ◽

Ribosome Formation

ABSTRACTIndividually, the ribosomal proteins L1, L23, L36 and S6 are not essential for cell proliferation ofB. subtilis, but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg2+content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+content improved the growth rate of the ΔrplA(L1) and the ΔrplW(L23) mutant but did not restore those of the ΔrpmJ(L36) and the ΔrpsF(S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion ofyhdPand overexpression ofmgtEincreased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA(L1) mutant. These results indicate that Mg2+can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCEWe previously reported that an increase in the cellular Mg2+content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36 and S6), an increase in the cellular Mg2+content could restore the 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

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Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change

Cell ◽

10.1016/j.cell.2018.02.005 ◽

2018 ◽

Vol 173 (1) ◽

pp. 196-207.e14 ◽

Cited By ~ 22

Author(s):

Jeffrey N. Carey ◽

Erin L. Mettert ◽

Manuela Roggiani ◽

Kevin S. Myers ◽

Patricia J. Kiley ◽

...

Keyword(s):

Environmental Change ◽

Signaling Network ◽

Bacterial Signaling ◽

Rapid Environmental Change

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Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change

Cell ◽

10.1016/j.cell.2018.11.051 ◽

2018 ◽

Vol 175 (7) ◽

pp. 1989-1990 ◽

Cited By ~ 7

Author(s):

Jeffrey N. Carey ◽

Erin L. Mettert ◽

Manuela Roggiani ◽

Kevin S. Myers ◽

Patricia J. Kiley ◽

...

Keyword(s):

Environmental Change ◽

Signaling Network ◽

Bacterial Signaling ◽

Rapid Environmental Change

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An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1416485112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E747-E756 ◽

Cited By ~ 110

Author(s):

TuAnh Ngoc Huynh ◽

Shukun Luo ◽

Daniel Pensinger ◽

John-Demian Sauer ◽

Liang Tong ◽

...

Keyword(s):

Practical Interest ◽

Stringent Response ◽

Adenosine Monophosphate ◽

Molecular Target ◽

Type I ◽

Structural Studies ◽

Chemical Proteomics ◽

Bacterial Signaling ◽

Hydrolysis Of ◽

Hydrolysis Activity

The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.

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