Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ϕ29 Appendage Protein GP12 Using Synthetic Substrate Analogs

Myers, Cullen L.; Ireland, Ronald G.; Garrett, Teresa A.; Brown, Eric D.

doi:10.1074/jbc.m115.662866

Cited by 14 publications

(11 citation statements)

References 54 publications

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“…GlpQ and PhoD Catalyze Distinct Modes of WTA Degradation-Recognizing that the heterogeneity of WTA isolated from cells hindered enzyme kinetic studies and characterization of degradation products, chemically defined synthetic analogs of WTA intermediates were employed as substrates. We prepared tridecane-containing analogs of the TagF biosynthetic product (lipid .40 analog) with 14 C incorporated into the GroP polymer, and monitored product formation after reactions with GlpQ or PhoD using high performance anion exchange chromatography (HPAEX) (15). GlpQ reaction products included GroP and species similar in apparent size to the starting substrate ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S2). It was also evident that many PhoD-generated products would be undetectable by PAGE based on apparent sizes less than 20 polymer units (15).…”

Section: Resultsmentioning

confidence: 99%

“…To resolve this ambiguity, we performed a substrate trapping experiment where degradation was initiated on the 14 C-lipid .40 analog, and then an excess amount of unlabeled competing substrate was added to the reaction to trap dissociated enzyme. For the latter, we used a poly(GroP) polymer prepared with CDP-Gro as the acceptor, denoted CMP-poly(GroP) (15). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Synthesis of WTA Intermediate Analogs-[ 14 C]GroP lipid .40 analog and CMP-poly(GroP) were synthesized as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…More recently, B. subtilis impaired in the transfer of WTA to PG, contrary to S. aureus likewise impaired (12), was shown not to release WTA into the extracellular medium or accumulate WTA intermediates in the cell envelope (13), possible signs of WTA turnover and recovery. Nevertheless, the only enzymes shown to catalyze WTA degradation are of bacteriophage origin, for example the tail-spike protein GP12 from B. subtilis bacteriophage .29 (14,15).…”

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confidence: 99%

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Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

Myers

Koo

et al. 2016

Journal of Biological Chemistry

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The cell wall of most Gram-positive bacteria contains equal amounts of peptidoglycan and the phosphate-rich glycopolymer wall teichoic acid (WTA). During phosphate-limited growth of the Gram-positive model organism Bacillus subtilis 168, WTA is lost from the cell wall in a response mediated by the PhoPR two-component system, which regulates genes involved in phosphate conservation and acquisition. It has been thought that WTA provides a phosphate source to sustain growth during starvation conditions; however, WTA degradative pathways have not been described for this or any condition of bacterial growth. Here, we uncover roles for the Bacillus subtilis PhoP regulon genes glpQ and phoD as encoding secreted phosphodiesterases that function in WTA metabolism during phosphate starvation. Unlike the parent 168 strain, ΔglpQ or ΔphoD mutants retained WTA and ceased growth upon phosphate limitation. Characterization of GlpQ and PhoD enzymatic activities, in addition to X-ray crystal structures of GlpQ, revealed distinct mechanisms of WTA depolymerization for the two enzymes; GlpQ catalyzes exolytic cleavage of individual monomer units, and PhoD catalyzes endo-hydrolysis at nonspecific sites throughout the polymer. The combination of these activities appears requisite for the utilization of WTA as a phosphate reserve. Phenotypic characterization of the ΔglpQ and ΔphoD mutants revealed altered cell morphologies and effects on autolytic activity and antibiotic susceptibilities that, unexpectedly, also occurred in phosphate-replete conditions. Our findings offer novel insight into the B. subtilis phosphate starvation response and implicate WTA hydrolase activity as a determinant of functional properties of the Gram-positive cell envelope.

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Section: Resultsmentioning

confidence: 99%

“…S2). It was also evident that many PhoD-generated products would be undetectable by PAGE based on apparent sizes less than 20 polymer units (15).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Synthesis of WTA Intermediate Analogs-[ 14 C]GroP lipid .40 analog and CMP-poly(GroP) were synthesized as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

Myers

Koo

et al. 2016

Journal of Biological Chemistry

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Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

Harvey

2021

Mass Spectrometry Reviews

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This review is the ninth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo‐ and poly‐saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.

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B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro

Gale

Sun

et al. 2017

Cell Chemical Biology

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Gram-positive bacteria endow their peptidoglycan with glycopolymers that are crucial for viability and pathogenesis. However, the cellular machinery that executes this function is not well understood. While decades of genetic and phenotypic work have highlighted the LytR-CpsA-Psr (LCP) family of enzymes as cell-wall glycopolymer transferases, their in vitro characterization has been elusive, largely due to a paucity of tools for functional assays. In this report, we synthesized authentic undecaprenyl diphosphate-linked wall teichoic acid (WTA) intermediates and built an assay system capable of monitoring LCP-mediated glycopolymer transfer. We report that all Bacillus subtilis LCP enzymes anchor WTAs to peptidoglycan in vitro. Furthermore, we probed the catalytic requirements and substrate preferences for these LCP enzymes and elaborated in vitro conditions for facile tests of enzyme function. This work sheds light on the molecular features of glycopolymer transfer and aims to aid drug discovery and development programs exploiting this promising antibacterial target.

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Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ϕ29 Appendage Protein GP12 Using Synthetic Substrate Analogs

Cited by 14 publications

References 54 publications

Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro

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