Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

Morlot, Cécile; Straume, Daniel; Peters, Katharina; Hegnar, Olav Aaseth; Simon, N. T.; Villard, Anne-Maria; Contreras‐Martel, Carlos; Leisico, Francisco; Breukink, Eefjan; Gravier‐Pelletier, Christine; Corre, Laurent Le; Vollmer, Waldemar; Pietrancosta, Nicolas; Håvarstein, Leiv Sigve; Zapun, André

doi:10.1038/s41467-018-05602-w

Cited by 37 publications

(30 citation statements)

References 65 publications

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“…Using two complementary approaches, the in vitro pulldown assay and the in vivo interaction assay, we established that DUF1727 is responsible for the interaction between the two proteins MurT and GatD and for complex formation. This observation was confirmed by recent crystallographic structures, where GatD docks to the C terminus of MurT (20,21). Our results, obtained using distinct approaches, showed that DUF1727 is sufficient for the interaction with GatD and, more importantly, is essential for complex activity.…”

Section: Discussionsupporting

confidence: 88%

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Gonçalves

Portela

Lobo

et al. 2019

Antimicrob Agents Chemother

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Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

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Section: Discussionsupporting

confidence: 88%

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Gonçalves

Portela

Lobo

et al. 2019

Antimicrob Agents Chemother

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“…In another example, the transcriptional regulator, whiA , which is involved in sporulation in Streptomyces ( Bush et al, 2013 ), appears to play a key role in the mycobacterial cell cycle, clustering with other components of cell division ( Figure 7—figure supplement 1C ). Furthermore, MSMEG_6276 – a putative mur ligase – clusters closely with the peptidoglycan synthesis protein, mviN ( Gee et al, 2012 ), and exhibits strong homology to murT/gatD from S. pneumoniae ( Morlot et al, 2018 ; Figure 7—figure supplement 1D ). In combination, these additional examples support the utility of image-based profiling for informing or validating hypothetical or predicted gene function – particularly those involved in cell-wall metabolism or DNA metabolism and cell-cycle regulation.…”

Section: Resultsmentioning

confidence: 99%

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Wet

Winkler

Mhlanga

et al. 2020

eLife

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Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.

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“…In those Gram‐positive species where D ‐ iso Gln is found at Position 2, D ‐Glu is enzymatically deamidated to D ‐ iso Gln by an enzyme complex of MurT/GatD . The structure of the MurT/GatD complex has been solved recently, revealing the mechanism of PG amidation by MurT, and also the mechanism through which GatD produces and channels the ammonia required for PG amidation to the MurT active site …”

Section: Pg Synthesismentioning

confidence: 99%

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

Booth

Lewis

2019

Protein Science

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Bacteria are surrounded by a complex cell envelope made up of one or two membranes supplemented with a layer of peptidoglycan (PG). The envelope is responsible for the protection of bacteria against lysis in their oft‐unpredictable environments and it contributes to cell integrity, morphology, signaling, nutrient/small‐molecule transport, and, in the case of pathogenic bacteria, host–pathogen interactions and virulence. The cell envelope requires considerable remodeling during cell division in order to produce genetically identical progeny. Several proteinaceous machines are responsible for the homeostasis of the cell envelope and their activities must be kept coordinated in order to ensure the remodeling of the envelope is temporally and spatially regulated correctly during multiple cycles of cell division and growth. This review aims to highlight the complexity of the components of the cell envelope, but focusses specifically on the molecular apparatuses involved in the synthesis of the PG wall, and the degree of cross talk necessary between the cell division and the cell wall remodeling machineries to coordinate PG remodeling during division. The current understanding of many of the proteins discussed here has relied on structural studies, and this review concentrates particularly on this structural work.

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Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

Cited by 37 publications

References 65 publications

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

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