A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis in Escherichia 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 pyrophosphate (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 interactions which, in combination with ΔybjG ΔlpxT ΔbacA, are lethal or reduce fitness. The screen uncovered novel connections to cell division, DNA replication/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. Loss of QseC causes overactivation of the QseB response regulator 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 QseC's absence and prior functions ascribed to QseBC may originate from cell wall defects. Altogether, our findings provide a framework for identifying new determinants of cell integrity that could be targeted in future therapies.

Keywords: BacA; QseC; bactoprenol; lysis; morphology; peptidoglycan.

Figure 1.. Strategy to screen for Δ3PP synthetic interactions.

(A) Δ3PP screen workflow. E. coli cells lacking three Und-PP phosphatases (ΔlpxT ΔybjG ΔbacA) and the lac operon (Lac -) harbor pbacA (i.e., pMAJ95), a derivative of the unstable mini-F plasmid expressing the lac operon and bacA in the presence of IPTG. pbacA is readily lost in this background and cells form white or sectored-blue colonies on media containing X-gal. Conversely, introducing synthetically sick or lethal mutant combinations (i.e., λ1098) leads to the retention of pbacA and formation of blue colonies on media containing X-gal. (B) Images depicting white colonies and sectored-blue colonies (left panel) or solid-blue colonies (right panel). The strains tested were MAJ876 (Δ3PP [parent]) and MAJ974 (Δ3PP ΔpgpB).

Figure 2.. Viability of Δ3PP mutant derivatives.

(A) pbacA readily segregates in the Δ3PP parent, resulting in white or sectored-blue colonies at 30°C, 37°C, and 42°C. (B and C) pbacA is retained in mutants that are synthetically lethal in the Δ3PP background and colonies appear solid-blue. Alternatively, the frequency of pbacA segregation is reduced in mutants that are synthetically sick in the Δ3PP background because pbacA confers a growth advantage. Above photographs: genotype and strain designation. Below photographs: fraction of white colonies to the total number of colonies. Additional Δ3PP mutant derivatives are shown in Figure S2 in the supplemental material.

Figure 3.. Morphology of Δ3PP mutant derivatives at 42°C.

Cells with the indicated genotypes were grown at 42°C in LB (-pbacA) or LB containing chloramphenicol and 500 μM IPTG (+pbacA) until the culture reached an OD₆₀₀ of 0.3–0.4. The cells were then photographed by phase-contrast microscopy. Images of Δ3PP mutants grown at 37°C are shown in Figure S4 in the supplemental material. The white bar represents 3 μm. (A) The Δ3PP parent. (B) Mutant derivatives that produce shape defects in the absence of bacA expression. (C) Mutant derivatives that produce shape defects independent of bacA expression. Data are representative of at least two independent experiments.

Figure 4.. New shape-determinants.

(A) Cells with the indicated genotypes were grown at 37°C in LB until the culture reached an OD₆₀₀ of 0.5. The cells were then photographed by phase-contrast microscopy. White arrows point to examples of branching. The white bar represents 3 μm. (B) Live cells from panel A were also examined by flow cytometry. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size of the wild type is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of two independent experiments. The strains shown are MAJ25 (WT), MAJ964 (ΔenvZ), MAJ938 (ΔompR), MAJ969 (Δgor), and MAJ1005 (ΔqseC).

Figure 5.. QseB overactivation disrupts cell shape.

(A) Schematic depicting activated QseC phosphorylating and activating QseB (wild type). PmrB and one or more unknown factors (?) phosphorylate QseB in the absence of QseC (ΔqseC). Unlike QseC, however, PmrB cannot efficiently dephosphorylate QseB, leading to excessive amounts of activated QseB (Guckes et al., 2013). (B) Cells with the indicated genotypes were grown at 42°C in LB until the culture reached an OD₆₀₀ of 0.5. The cells were then photographed by phase-contrast microscopy. The white bar represents 3 μm. (C) Live cells from panel B were also examined by flow cytometry. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size of the wild type is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of two independent experiments. The strains shown are MAJ25 (WT), MAJ1002 (ΔpmrB), MAJ962 (ΔqseB), MAJ1005 (ΔqseC), MAJ1003 (ΔpmrB ΔqseC), and MAJ999 (ΔqseBC).

Figure 6.. Suppression of ΔqseC shape defects.

(A) Workflow of strategy to uncover multicopy plasmids that suppress the shape defect of ΔqseC cells. Δ3PP ΔqseC cells harboring pbacA produce predominantly blue colonies at 42°C (cf., Figure 2C). However, pbacA segregates in cells containing plasmids (pORF) that suppress the ΔqseC shape defect, and appear light blue (i.e., sectored-blue). Note that white colonies (indicating loss of pbacA) always yielded misshapen cells. (B) Micrographs of ΔqseC cells containing derivatives of pBRplac that express qseC or the small RNA rprA. Cells with the indicated genotypes were grown at 42°C in LB until the culture reached an OD₆₀₀ of 0.5. The cells were then photographed by phase-contrast microscopy. The white bar represents 3 μm. (C) Live cells from panel B were also examined by flow cytometry. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size of the wild type is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of two independent experiments. The strains shown are MAJ1177 (pqseC), MAJ1143 (vector), MAJ1144 (—), MAJ1151 (ΔrpoS), and MAJ1163 (Δhfq/prprA).