Stabilization of G Domain Conformations in the tRNA-modifying MnmE-GidA Complex Observed with Double Electron Electron Resonance Spectroscopy

Böhme, Sabine; Meyer, Simon De; Krüger, André; Steinhoff, Heinz‐Jürgen; Wittinghofer, Alfred; Klare, Johann P.

doi:10.1074/jbc.m109.096131

Cited by 30 publications

(28 citation statements)

References 37 publications

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“…It has been shown that GidA potentially regulates bacterial factors by a post-transcriptionally based mechanism to modify tRNA (Yim et al 2006;Moukadiri et al 2009;Shi et al 2009;Bohme et al 2010;Shippy et al 2011). Specifically, in E. coli, GidA (also called MnmG) forms a complex with MnmE that catalyzes the addition of a carboxymethylaminomethyl group at the 5 position of the wobble uridine (U34) of tRNAs (Elseviers et al 1984;Bregeon et al 2001;Meyer et al 2009).…”

Section: Discussionmentioning

confidence: 99%

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium

et al. 2011

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Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

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Section: Discussionmentioning

confidence: 99%

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium

et al. 2011

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“…GidA was originally thought to be involved in cell division due to the filamentous morphology observed when the cells were grown in rich medium supplemented with glucose [13]. More recent studies done in E. coli have shown GidA modulates several bacterial factors by a post-transcriptional mechanism to modify tRNA by the addition of a cmnm group at the 5 position of the wobble uridine (U34) of tRNAs [15-19,30,31]. It has been proposed that tRNA modification can serve as a regulatory mechanism to modulate gene expression[32].…”

Section: Discussionmentioning

confidence: 99%

Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine

Shippy

Fadl

2012

BMC Microbiology

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BackgroundSalmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine.ResultsThe protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Naïve mice receiving either cells or sera from immunized mice were partially protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-γ, and IL-10 cytokines in splenocytes induced with STM cell lysate.ConclusionsTogether, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.

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“…The MnmE G-domain is relatively far from the active centre of the MnmEG complex. However, previous findings have indicated that interactions of MnmE with MnmG on a site remote from the MnmE G-domain co-stimulate GTP hydrolysis (18,49). Therefore, it is possible that tRNA binding to the MnmEG active centre might modulate the GTPase cycle.…”

Section: Discussionmentioning

confidence: 91%

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

Díaz‐Prado

Villarroya

Medina

et al. 2013

Nucleic Acids Research

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MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.

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Stabilization of G Domain Conformations in the tRNA-modifying MnmE-GidA Complex Observed with Double Electron Electron Resonance Spectroscopy

Cited by 30 publications

References 37 publications

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium

Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

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