title: ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.
abstract: 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 Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive 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.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom; a.grundling@imperial.ac.uk r.corrigan@sheffield.ac.uk.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Corrigan
authors.affiliation: Section of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Bellows
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom;
authors.forename: Alison
authors.initials: A
authors.lastname: Wood
authors.affiliation: Section of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom a.grundling@imperial.ac.uk r.corrigan@sheffield.ac.uk.
authors.forename: Angelika
authors.identifier: http://orcid.org/0000-0002-6235-8687
authors.initials: A
authors.lastname: Gründling
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000378:metabolism; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D015723:Gene Library; D006159:Guanosine Tetraphosphate / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000096:biosynthesis; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests; D016366:Open Reading Frames; D001678:Organelle Biogenesis; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006159:Guanosine Tetraphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases
keywords: Staphylococcus aureus; ppGpp; ribosome; stringent response; tolerance