A Bacterial G Protein-Mediated Response to Replication Arrest

Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The ⌬nos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the ⌬nos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.D. radiodurans ͉ nitric oxide synthase ͉ UV radiation

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Endogenous nitric oxide regulates the recovery of the radiation-resistant bacterium Deinococcus radiodurans from exposure to UV light

Patel

Moreau

Widom

et al. 2009

“…Only one of these proteins, ObgE from E. coli, is known to interact with (p)ppGpp (56). ObgE has been implicated in DNA replication (60) and has also been shown to bind to the Gramnegative (p)ppGpp synthetase/hydrolase enzyme SpoT from E. coli (61). Similar to RsgA, RbgA, HflX, and Era, it has recently been shown that ObgE also has a role in 50S and 30S ribosomal subunit association, and that (p)ppGpp binding can enhance the association of ObgE to the 50S subunit of the ribosome (59).…”

Section: Discussionmentioning

confidence: 99%

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

Corrigan

Bellows

Wood

et al. 2016

180

227

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

“…All strains are isogenic with MG1655 (F Ϫ rph-1) (33). The iraD::Tn5 Insertion mutant, STL9197, was isolated by electroporation of the EZ-Tn5 ϽR6K ori KAN-2Ͼ transpososome complex (Epicentre) into MG1655 (34); all other strains were constructed by P1 transduction (35). Cultures were grown at 37°C in LB supplemented with appropriate antibiotics at 20 g/mL kanamycin, 100 g/mL ampicillin, and/or 10 g/mL tetracycline.…”

Section: Methodsmentioning

confidence: 99%

“…Treatment was terminated with catalase (Sigma-Aldrich, from Aspergillus niger, 180 ng/mL), and cell survival was determined by serial dilution and plating. Plating efficiency assays in the presence of AZT were done as described (34); phleomycin (Invitrogen) treatment was performed similarly with doses of 0.5-0.65 g/mL.…”

Section: Methodsmentioning

confidence: 99%

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

Merrikh

Ferrazzoli

Bougdour

et al. 2009

Self Cite

We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.oxidative stress ͉ posttranslational regulation ͉ replication stress ͉ SOS response ͉ DNA repair T hroughout its life cycle, Escherichia coli is faced with different environmental challenges and regulates gene expression accordingly. One way is by changes in the promoter recognition of RNA polymerase via different situation-specific factors (1). In E. coli, the major alternative sigma factor is S (RpoS), which is required for expression of specific genes on entry to stationary phase or as a response to stress (2-4). Although the RpoS dependence of many of these responses and the regulation of RpoS itself have been well studied, the relevance of this to DNA repair has not been a major focus.To find genes important in DNA damage responses, we performed a random Tn5 transposon insertion mutant screen, assaying sensitivity to, among other agents, phleomycin and azidothymidine (AZT). Phleomycin induces random single-or double-strand breaks in the backbone of DNA (5), whereas AZT blocks DNA synthesis, leading to single-strand gaps in the replication fork (6). One insertion mutant in iraD (previously an unknown gene, yjiD) was hypersensitive to phleomycin and AZT.Recent work from Gottesman and coworkers (7) implicated IraD in posttranslational regulation of RpoS. The RssB adaptor protein targets RpoS to ClpXP for degradation during logarithmic growth, keeping RpoS protein levels low in the absence of stress (8-12). IraD was identified in a high-copy plasmid screen for genes promoting accumulation of an RpoS-LacZ fusion protein. The IraD gene product acts as an antiadaptor protein via direct binding and inhibition of the ability of RssB to target RpoS for proteolysis by ClpXP in vitro (7).In the work presented here, we demonstrate that IraD is required for survival to DNA damage, providing evidence of the physiological importance of IraD in particular and the antiadaptor mechanism in general. The data presented suggest that IraD acts as an antagonist of RssB, regulating RpoS levels and stabilization, not only after DNA damage but constitutively. Our results establish the importance of RpoS stabilization in proliferating bacterial cells in which replication has been direc...