Carbonyl reduction by YmfI in <i>Bacillus subtilis</i> prevents accumulation of an inhibitory EF‐P modification state

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

doi:10.1111/mmi.13760

Cited by 30 publications

(29 citation statements)

References 39 publications

(65 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Further, we showed that 5-aminopentanone, but not unmodified EF-P, is inhibitory to swarming motility, as abolishing posttranslational modification of EF-P altogether by mutation of Lys32 to an arginine suppressed the ymfI swarming defect ( 24 ). Here, we take advantage of EF-P activity in the absence of modification to identify other enzymes that act upstream of YmfI in the EF-P modification pathway ( 24 ). Mass spectrometry analyses of EF-P purified from wild-type (WT) B. subtilis and each of the modification mutants revealed that EF-P can retain incomplete modifications.…”

Section: Introductionmentioning

confidence: 96%

“…We have recently shown that YmfI reduces 5-aminopentanone to 5-aminopentanol in the final step of EF-P modification ( 24 ). Further, we showed that 5-aminopentanone, but not unmodified EF-P, is inhibitory to swarming motility, as abolishing posttranslational modification of EF-P altogether by mutation of Lys32 to an arginine suppressed the ymfI swarming defect ( 24 ). Here, we take advantage of EF-P activity in the absence of modification to identify other enzymes that act upstream of YmfI in the EF-P modification pathway ( 24 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Witzky

Hummels

Tollerson

et al. 2018

mBio

Self Cite

View full text Add to dashboard Cite

Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In Bacillus subtilis, EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that ynbB, gsaB, and ymfI are required for modification and that yaaO, yfkA, and ywlG influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the efp mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the efp mutant or wild-type B. subtilis depending on the growth condition. In vivo polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence.

show abstract

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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

Witzky

Hummels

Tollerson

et al. 2018

mBio

Self Cite

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

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

Liu

et al. 2019

J Bacteriol

View full text Add to dashboard Cite

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.

show abstract

“…The peptidase U32 protein YrrN is one member of the yrrMNO operon which is important for the biosynthesis of 5-hydroxyuridine, YrrN is involved in the synthesis of 5methoxyuridine (Nguyen et al 2011). The cytoplasmic ClpP-like germination protease TepA is involved in spore outgrowth in B. subtilis, and is not proposed to be a signal peptide peptidase but to degrade a specialized family of small DNA-binding proteins during the process of spore outgrowth (Traag et al 2013;Westers et al 2004a).YmfH is identi ed by the presence of Peptidase_M16 and Peptidase_M16_C domains, and is predicted to encode uncharacterized zinc protease with unknown speci city (Hummels et al 2017).In this study, the deletion of any of the four proteases improved the secretion of AiiO-AIO6, but the degree of improvement was different. Their effect on the secretion of AiiO-AIO6 in B. subtilis was YwpE > YrrN > TepA or YmfH, which may be due to the difference in the degradation ability of these proteases to AiiO-AIO6 or the degradation ability of the proteins assisting the secretion of AiiO-AIO6.…”

Section: Discussionmentioning

confidence: 99%

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficientBacillus subtilis

Xia

Yang

Pan

et al. 2020

Preprint

View full text Add to dashboard Cite

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway.To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrNandywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrNand BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

show abstract

Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF‐P modification state

Cited by 30 publications

References 39 publications

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

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

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

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficientBacillus subtilis

Contact Info

Product

Resources

About