Structural and Biochemical Analysis of the Obg GTP Binding Protein

Buglino, John A.; Shen, Vincent K.; Hakimian, P.; Lima, Christopher D.

doi:10.1016/s0969-2126(02)00882-1

Cited by 161 publications

(254 citation statements)

References 40 publications

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“…Consistent with the data from E. coli, CgtA showed an interaction with SpoT. There was also self-interaction, consistent with the data showing CgtA-SpoT interaction in E. coli and also consistent with structural data showing that CgtA is found as a dimer in B. subtilis and Thermus thermophilus (32,33). CgtA did not interact with RelA, despite significant sequence similarity between RelA and SpoT in V. cholerae (32% identity/51% similarity).…”

Section: Cgta Depletion Causes An Increase In Intracellular Concentrasupporting

confidence: 89%

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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Section: Cgta Depletion Causes An Increase In Intracellular Concentrasupporting

confidence: 89%

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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“…Protein Expression and Purification-Expression and purification of B. subtilis Obg and Hemophilus influenzae YchF was performed as described elsewhere (14,16). Expression of all other proteins was done in E. coli BLR (pRep4).…”

Section: Methodsmentioning

confidence: 99%

“…The affinity of hOLA1 for ATP and the intrinsic ATP hydrolysis rate are comparable with the GTP binding and hydrolysis properties of other Obg family members (28 -30). hOLA1 might therefore constitute a novel group of ATPases, with biochemical features resembling those of related Obg-like low affinity GTPases (16,28,29).…”

Section: Hola1 An Obg-like Atpasementioning

confidence: 99%

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Human OLA1 Defines an ATPase Subfamily in the Obg Family of GTP-binding Proteins

Koller-Eichhorn

Marquardt

Gail

et al. 2007

Journal of Biological Chemistry

100

140

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Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.

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“…In Gram-negative bacteria, this transcriptional shift is mediated by (p)ppGpp interacting with the RNA polymerase (RNAP), which in combination with the transcription factor DksA, modulates gene expression on a transcriptional level (3,14). Aside from the RNAP, there are at least 15 other direct (p)ppGpp target proteins in Escherichia coli, such as the translation elongation factors EFG and EF-Tu, the DNA primase DnaG, and a number of amino acid decarboxylases that are involved in the acid stress response (15)(16)(17). These nucleotides also play major roles in controlling bacterial persistence in Gram-negative bacteria by activating toxin-antitoxin systems and triggering slow growth (9), leading to cells that persist in the host following antibiotic treatment.…”

mentioning

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.

185

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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Structural and Biochemical Analysis of the Obg GTP Binding Protein

Cited by 161 publications

References 40 publications

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

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

Human OLA1 Defines an ATPase Subfamily in the Obg Family of GTP-binding Proteins

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

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