EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in
            <i>Bacillus subtilis</i>

Witzky, Anne; Hummels, Katherine R.; Tollerson, Rodney; Rajkovic, Andrei; Jones, Lisa; Kearns, Daniel B.; Ibba, Michael

doi:10.1128/mbio.00306-18

Cited by 32 publications

(36 citation statements)

References 29 publications

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“…The function of both EF-P and e/aIF-5A in assisting polyproline translation depends on posttranslational modification (8,14). In this regard, EF-P can be ␤-lysylated (15)(16)(17), 5-aminopentolylated (18)(19)(20), and rhamnosylated (7,21,22). The eukaryotic/archaeal ortholog e/aIF5A, in contrast, strictly depends on (deoxy)hypusination (23).…”

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confidence: 99%

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Liu

et al. 2019

J Bacteriol

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A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-β-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species. IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

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confidence: 99%

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Liu

et al. 2019

J Bacteriol

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“…Modification status of other bacteria is unknown. Highly possible modification of S.aureus EF-P could be 5-aminopentanolitation of 34 K, the same type modification as for B.subtillus EF-P [15].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore the knowledge about dynamics of the loop I could be crucial for understanding of EF-P interaction process with P-tRNA near PTC and the function of modifications. Recently we analyzed a dynamic of SaEF-P backbone amide protons by high-resolution NMR spectroscopy [17] and due to high mobility of the loop between β-sheets β2 and β3 (residues 30-34) the amide protons have not been detected in 15 N-1 H heteronuclear NMR spectra due to fast proton exchange leading to the absence of the corresponding amide resonances. Site-directed spin labeling in concert with electron paramagnetic resonance (EPR) spectroscopy is a powerful method for studying the nature of proteins in solution which has been especially useful for examining proteins that are large, flexible or highly dynamic [18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Structural dynamics of a spinlabeled ribosome elongation factor P (EF-P) from Staphylococcus aureus by EPR spectroscopy

et al. 2019

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Elongation factor P (EF-P) is a three domain protein that binds to the ribosome between P and E sites.The EF-P involved in a specialized translation of stalling amino acid motifs such as (PPP or APP). Proteins with stalling motifs are involved in various cell processes, including stress resistance and virulence of bacteria. EF-P stabilizes the P-tRNA and increases the entropy of stalled ribosome complex to compensate for rigid nature of proline residue, thus providing adequate protein synthesis rate. Detailed structural mechanisms of this effect are still poorly understood. It was shown that most of bacteria needs a special post-translational modification in a conservative region of the loop in the domain I of the EF-P located near the CCA-end of P-tRNA and the peptidyl transferase center. In present paper by EPR spectroscopy we investigated a spinlabeled EF-P from Staphylococcus aureus-a pathogenic bacteria which causes various human diseases. Addition of MTSL ((S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)) label covalently bound to 32Cys in the highly conservative part of EF-P loop in the domain I of the EF-P allows us to analyze protein dynamic by EPR spectroscopy of the high mobility region which was not observed in NMR spectra due to fast proton exchange leading to the absence of the corresponding amide NMR resonances. We suppose that our result could be extended in future for the analysis of EF-P-ribosome complex formation process by advanced EPR techniques.

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“…While EF-P ␤-lysylation was originally described in enterobacteria, it is now clear that several other posttranslational modification pathways exist in more divergent bacteria. A lysine residue is posttranslationally modified in Bacillus subtilis by a range of enzymes (15). In Pseudomonas aeruginosa, EF-P is instead rhamnosylated at a conserved arginine residue by EarP (16).…”

mentioning

confidence: 99%

“…In Pseudomonas aeruginosa, EF-P is instead rhamnosylated at a conserved arginine residue by EarP (16). Unlike most species in which mutations to modifying enzymes produce a similar phenotype to efp mutants, B. subtilis-modifying enzyme mutants show distinct phenotypes, including altered proline translation rate and sensitivity to certain chemicals (15).…”

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confidence: 99%

Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora

et al. 2019

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Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the β-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or β-lysylated EF-P. IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.

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EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis

Cited by 32 publications

References 29 publications

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Structural dynamics of a spinlabeled ribosome elongation factor P (EF-P) from Staphylococcus aureus by EPR spectroscopy

Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora

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