A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis inEscherichia coli

Abstract

Undecaprenyl phosphate (Und-P) is an essential lipid carrier that ferries cell wall intermediates across the cytoplasmic membrane in bacteria. Und-P is generated by dephosphorylating undecaprenyl diphosphate (Und-PP). In Escherichia coli, BacA, PgpB, YbjG, and LpxT dephosphorylate Und-PP and are conditionally essential. To identify vulnerabilities that arise when Und-P metabolism is defective, we developed a genetic screen for synthetic mutations in combination with ΔybjG ΔlpxT ΔbacA. The screen uncovered system-wide connections, including novel connections to cell division, DNA replication and repair, signal transduction, and glutathione metabolism. Further analysis revealed several new morphogenes; loss of one of these, qseC, caused cells to enlarge and lyse. QseC is the sensor kinase component of the QseBC two-component system. In the absence of QseC, the QseB response regulator is overactivated by PmrB cross-phosphorylation. Here, we show that deleting qseB completely reverses the shape defect of ΔqseC cells, as does overexpressing rprA (a small RNA). Surprisingly, deleting pmrB only partially suppressed qseC-related shape defects. Thus, QseB is activated by multiple factors in the absence of QseC and functions ascribed to QseBC may be related to cell wall defects. Altogether, our findings provide a framework for identifying new determinants of cell integrity that could be targeted in future therapies.

Genetic interaction mapping highlights key roles of the Tol‐Pal complex

“…genes (Jorgenson & Bryant, 2021), consistent with our idea that tol-pal strains are sensitive to cell wall defects linked to und-P sequestration.…”

“…Generally, Δ tolQ and Δ tolA mutants appeared less tolerant of disruption in genes involved in two major processes, namely cell wall biosynthesis/remodeling and OM assembly/integrity (Table 1). For cell wall biosynthesis/remodeling, we specifically found genes involved in peptidoglycan synthesis ( dapF , lpoA , mrcA , lpoB , mrcB ), remodeling ( nlpI , prc , tatC ), recycling ( ldcA ), and undecaprenyl lipid carrier sequestration ( qseC , wecB , wecC , wecD , wecE , wecF , wzxE [Jorgenson & Bryant, 2021; Jorgenson et al, 2016]); for OM assembly/integrity, genes involved in LPS synthesis/modification ( gtrS , waaP , waaG ), β‐barrel protein assembly ( bamB , degP , surA ), and OM‐cell wall tethering ( ompA ) exhibited negative interactions with tol‐pal . We successfully constructed clean deletions of each gene in the Δ tolA background, with the exception of qseC , where a depletion strain was constructed (Figure S5).…”

“…One of the strongest negative genetic interaction with tolQ and tolA is qseC , encoding the sensor kinase of the QseBC two‐component system. This system has known regulatory roles in quorum sensing, motility (Sperandio et al, 2002), and polymyxin tolerance (Guckes et al, 2017), but was recently discovered in a genetic screen to also be important for the maintenance of cell morphology and for survival when und‐P is limited (Jorgenson & Bryant, 2021). While the mechanism remains to be clarified, the synthetic defects observed between Δ tolQ/A and qseC are therefore likely related to problems in cell wall and/or a limited und‐P pool.…”

Genetic interaction mapping highlights key roles of the Tol-Pal complex

“…This is not unexpected given that the build-up of ECA-related intermediates is known to result in cell wall defects via the sequestration of undecaprenyl-phosphate (und-P) [32], the lipid carrier also used in peptidoglycan biosynthesis; indeed, wec tol double mutants do display more severe morphological defects than the corresponding single mutants [26]. One of the strongest negative genetic interaction with tolQ and tolA is qseC, encoding the sensor kinase of the QseBC two-component system, that has recently been discovered to be important for cell wall homeostasis, especially when und-P pool is limited [31]. Interestingly, the same genetic screen for mutants sensitive to a limited und-P pool also identified wec and tol-pal genes, overall suggesting that tol-pal strains are sensitive to cell wall defects linked to und-P sequestration.…”

“…Generally, ΔtolQ and ΔtolA mutants appeared less tolerant of disruption in genes involved in two major processes, namely cell wall biosynthesis/remodeling and OM assembly/integrity (Table 1). For cell wall biosynthesis/remodeling, we specifically found genes involved in peptidoglycan synthesis (dapF, lpoA, mrcA, lpoB, mrcB), remodeling (nlpI, prc, tatC), recycling (ldcA), and undecaprenyl lipid carrier sequestration (qseC, wecB, wecC, wecD, wecE, wecF, wzxE [31,32]); for OM assembly/integrity, genes involved in LPS synthesis/modification (gtrS, waaP, waaG), β-barrel protein assembly (bamB, degP, surA), and OM-cell wall tethering (ompA) exhibited negative interactions with tol-pal. We successfully constructed clean deletions of each gene in the ΔtolA background, with the exception of qseC, where a depletion strain was constructed (Fig.…”