Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

Abstract

The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the M -dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the M regulon to cell envelope homeostasis pathways. IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

High‐throughput CRISPRi phenotyping identifies new essential genes inStreptococcus pneumoniae

“…Indeed, by sequencing the two key elements of the CRISPRi system, the sgRNA and dcas9 , we found that most of the suppressor strains contain loss‐of‐function mutation in the dcas9 coding sequence (Fig EV4B). This is similar to observations made for the CRISPRi system in Bacillus subtilis (Zhao et al , ).…”

Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis

“…Cells lacking r M are significantly more sensitive to drugs that interrupt essential steps in the construction of PG, which is at least partially due to an inability to induce genes involved in PG homeostasis (Mascher et al, 2007;Luo and Helmann, 2012). Although the roles of many genes in the r M regulon have been well defined (Cao et al, 2005;Eiamphungporn and Helmann, 2008;Meeske et al, 2015;Meeske et al, 2016;Zhao et al, 2016), the contributions of each r M target operon to antibiotic resistance are incompletely understood. Here, we show that antibiotic-dependent induction of the Spx regulon protects cells against killing by cell wall antibiotics (Fig.…”

Aspartate deficiency limits peptidoglycan synthesis and sensitizes cells to antibiotics targeting cell wall synthesis in Bacillus subtilis

“…LFH PCR, chromosomal and plasmid DNA transformation was performed as previously stated (Zhao et al, ). The kanamycin resistance and MLS resistance cassettes were PCR amplified from plasmids pDG780 and pDG646 respectively (Guerout‐Fleury et al, ).…”