d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Fura, Jonathan M.; Kearns, Daniel B.; Pires, Marcos M.

doi:10.1074/jbc.m115.683342

Cited by 56 publications

(63 citation statements)

References 45 publications

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“…Although penicillin-binding proteins and Ldts are evolutionary and structurally distinct transpeptidases, research in diverse bacteria showed that both enzyme types can exchange a range of naturally occurring D-amino acids (DAAs) with the fifth- and fourth-position D-alanines in the peptide stems of PG subunits, respectively 2–4 (Fig. 1b).…”

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confidence: 99%

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation

et al. 2017

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Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

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Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation

et al. 2017

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“…The peptide portion of the peptidoglycan biopolymer terminates in two D-alanine residues. Derivatives of D-alanine and D-alanine-D-alanine have been used in many bacterial species, including mycobacteria, to detect cell wall metabolism (Botella et al, 2017;Fura et al, 2015;Hayashi et al, 2018;Kuru et al, 2012;Liechti et al, 2014;Lebar et al, 2014;Meniche et al, 2014;Siegrist et al, 2013;Siegrist et al, 2015). Single residue D-amino acid probes have been hypothesized to incorporate into peptidoglycan in part or in whole via periplasmic transpeptidases (Siegrist et al, 2015).…”

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confidence: 99%

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

et al. 2018

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In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

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“…One possible labeling strategy involves using penicillin-binding proteins (PBPs) to exchange the terminal D-alanine on the Lipid II stem peptide with D-amino acids bearing isotope labels, fluorophores, or functional groups that allow derivatization (Figure 1b). 4 To implement this strategy, suitable enzymes must be identified.…”

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Identification of a Functionally Unique Family of Penicillin-Binding Proteins

et al. 2017

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Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high molecular weight PBPs, which are transpeptidases that form peptidoglycan crosslinks, and low molecular weight PBPs, which are typically hydrolases. We report a functionally unique family of low molecular weight PBPs that act as transpeptidases rather than hydrolases, but they do not crosslink peptidoglycan. We show that these PBPs can exchange D-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into peptidoglycan and, further, that this linkage can occur via attack of the primary ε-amine side chain, an unprecedented reaction for a PBP.

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d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Cited by 56 publications

References 45 publications

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

Identification of a Functionally Unique Family of Penicillin-Binding Proteins

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