The<i>Escherichia coli</i>GTPase CgtA<sub>E</sub>Cofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

Wout, Patrice K.; Pu, Krishnaraj; Sullivan, Susan M.; Reese, Virginia; Zhou, Shaoman; Lin, Bin; Maddock, Janine R.

doi:10.1128/jb.186.16.5249-5257.2004

Cited by 138 publications

(207 citation statements)

References 67 publications

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“…The cell lysates were separated by sucrose density gradient centrifugation, and GTP-binding proteins in each fraction were identified by immunoblotting with specific antibodies. Obg was detected in fractions containing the free 50 S subunit in the presence of the nonhydrolyzable GTP analog, GTP␥S (data not shown), as reported previously (17,18). In addition we newly found that YlqF stably co-sedimented with the free 50 S subunit in the presence of GTP␥S, but not GTP or GDP (Fig.…”

Section: Resultssupporting

confidence: 71%

“…YlqF Is Specifically Associated with the 50 S Ribosomal Subunit-Era, Obg, and YjeQ interact with ribosomal subunits in various bacteria (17)(18)(19)(20)(21)(22)27). Accordingly, we examined whether the essential GTPbinding proteins (Era, Obg, YsxC, YlqF, YphC, and YqeH) in B. subtilis are associated with the ribosome.…”

Section: Resultsmentioning

confidence: 99%

“…Obg and Era are the most extensively characterized bacterial small GTP-binding proteins belonging to this family. Obg interacts with the 50 S subunit in E. coli and Caulobacter crescentus (17,18). In Bacillus subtilis, Obg cofractionates with the 50 S subunit and specifically interacts with the ribosomal protein, L13 (19).…”

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confidence: 99%

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

Matsuo

Morimoto²,

Kuwano

et al. 2006

Journal of Biological Chemistry

111

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Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

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

Matsuo

Morimoto²,

Kuwano

et al. 2006

Journal of Biological Chemistry

111

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“…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

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

188

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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“…In B. subtilis, Obg interacts with the general stress response pathway and is necessary for activation of the stress resistance transcription factor, B , which is induced by both nutritional and physical stress (23). In E. coli, CgtA interacts with SpoT (24).…”

mentioning

confidence: 99%

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

Raskin

Judson²,

Mekalanos³

2007

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

107

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The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress response or ''stringent'' response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the stringent response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA؉ CgtA؉ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the stringent response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.alarmone ͉ cholera ͉ GTPase ͉ starvation ͉ transcription

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TheEscherichia coliGTPase CgtA_ECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

Cited by 138 publications

References 67 publications

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

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

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

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

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TheEscherichia coliGTPase CgtAECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

Cited by 138 publications

References 67 publications

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

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

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

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

Contact Info

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TheEscherichia coliGTPase CgtA_ECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase