The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli

Silva, Monton; Otten, Christian; Biboy, Jacob; Breukink, Eefjan; VanNieuwenhze, Michael S.; Vollmer, Waldemar; Blaauwen, Tanneke den

doi:10.3389/fmicb.2018.02101

Cited by 26 publications

(30 citation statements)

References 67 publications

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“…Combined with the recent findings of other groups (Peters et al, 2016b; Castanheira et al, 2017; Montón Silva et al, 2018), our results suggest that the active cell wall synthesis machinery varies across pH environments. We hypothesized that one potential consequence of environmental plasticity in the cell wall synthesis machinery may be changes in intrinsic resistance to cell wall active antibiotics.…”

Section: Resultssupporting

confidence: 88%

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

Mueller

Egan

Breukink

et al. 2019

eLife

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Although the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called ‘redundant’ enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1a and PBP1b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range and influences pH-dependent changes in resistance to cell wall active antibiotics. Altogether, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches. This specialization may ensure robust growth and cell wall integrity in a wide range of conditions.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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Section: Resultssupporting

confidence: 88%

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

Mueller

Egan

Breukink

et al. 2019

eLife

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“…These results suggest that the major mode(s) of FDAA incorporation in actively growing E. coli is due to the L , D -TPases, primarily LdtD. 41…”

Section: Resultsmentioning

confidence: 83%

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis

et al. 2019

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Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

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“…Adding to the complexity of this regulation, FtsW and CpoB, via LpoB, also suppress the activity of PBP1B (26, 27). However, at midcell, CpoB is likely removed from PBP1B by TolA, allowing PBP1b to become much more active (26, 28).…”

Section: Commentarymentioning

confidence: 99%

Checks and Balances in Bacterial Cell Division

Blaauwen

Luirink

2019

mBio

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Assembly of the division machinery in Gram-negative and Gram-positive bacteria occurs in two time-dependent steps. First, the FtsZ proto-ring localizes at midcell including some FtsN molecules. Subsequently, the proteins that catalyze and regulate septal peptidoglycan (PG) synthesis are recruited including among others, the FtsBLQ-PB1B-FtsW-PBP3 complex. Further accumulation of FtsN finally allows initiation of cell division. It was known that FtsA and FtsQLB somehow prevented this initiation. Recently, A. Boes, S. Olatunji, E. Breukink, and M. Terrak (mBio 10:e01912-18, 2019, https://doi.org/10.1128/mBio.01912-18) reported that this is caused by inhibition of the activity of the PG synthases by FtsBLQ, which has to be outcompeted by accumulation of the PBP1b activating FtsN. This supports a central structural as well as regulatory role for the FtsBLQ protein complex that is conserved only in prokaryotes, making it an attractive target for antibiotic development.

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The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli

Cited by 26 publications

References 67 publications

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis

Checks and Balances in Bacterial Cell Division

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