Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK

The role of PPK in the regulation of lipid A modification and polymyxin resistance.

PolyP synthesized by PPK upon a switch from LB to MOPS media triggers BasS activation by autophosphorylation. Activated BasS then transphosphorylases BasR to induce downstream transcription of the arnBCADTEF operon and EptA gene. This results in increased level of Arn and EptA proteins, and up-regulation of the respective L-Ara4N and pEtN modifications. Dashed arrows indicate an additional step where the modified lipid A (a key structural component of the LPS) is transported to the outer membrane by the LPS transport system. This reduces the net negative charge of the outer membrane and results in polymyxin B (PMB) resistance. What is still unknown is whether polyP is acting directly in BasS activation and if PPK has a role independent of polyP synthesis. (TIF)</p

Uncropped and unadjusted western blot, ponceau S, polyP gel, lipid A profile, and spot test images.

Uncropped and unadjusted western blot, ponceau S, polyP gel, lipid A profile, and spot test images.</p

Underlying data for qPCR analysis presented in Fig 3G.

Underlying data for qPCR analysis presented in Fig 3G.</p

Arn expression is PPK-dependent during MOPS starvation.

(A) Rescue of Arn expression following 3 h in MOPS media. Extracted protein samples were resolved using SDS-PAGE, transferred to PVDF, and detected using an anti-Flag antibody. (TIF)</p

Molecular control of Arn and EptA protein expression by PPK.

(A) Induction of ArnC-3Flag expression upon the switch from LB to MOPS media. The indicated strains were grown in LB media to mid-log phase and shifted to MOPS media for 3 h. Proteins were extracted and resolved via SDS-PAGE prior to transfer to a PVDF membrane. Tagged proteins were detected using an anti-Flag antibody. (B) Expression of PhoP between wild-type cells and Δppk mutants. The indicated strains were starved in MOPS media for 3 h prior to protein extraction, separation by SDS-PAGE, and detection with an antibody against PhoP. A background band (*) in ΔphoP mutants (controls used to validate the antibody) is PPK-regulated, which makes evaluation of changes to PhoP protein expression difficult. Regardless, regulation of PhoP by PPK appears to be minimal. (C) Expression of PhoQ-3Flag between wild-type cells and Δppk mutants. The indicated strains were starved in MOPS media for 3 h and proteins were analyzed as described in (B) using an antibody towards Flag. (D) Influence of magnesium (Mg2+) on ArnC-3Flag expression in MOPS media. Cells were grown to mid-exponential phase in LB and then shifted to MOPS minimal media in the absence or presence of 1 mM magnesium chloride or calcium chloride (control) for 3 h. Extracted protein samples were resolved using SDS-PAGE, transferred to PVDF, and detected using an anti-Flag antibody. Images shown are representative of results from ≥3 experiments. (E) Expression of RpoS between wild-type cells and Δppk mutants. The indicated strains were starved in MOPS media for 3 h prior to protein extraction, separation by SDS-PAGE, and detection with an antibody directed against RpoS. The ΔrpoS mutant strains serve to validate the antibody. (F) Expression of BasS-3Flag, BasR-3Flag, and ArnA-3Flag in LB supplemented with iron by wild-type and Δppk mutants. The indicated strains grown in LB or LB + iron (200 μm FeSO4) for 1.5 h prior to protein extraction, separation by SDS-PAGE, transfer to PVDF, and detection of tagged proteins with an anti-Flag antibody. (G, H) Influence of iron (G) and phoB mutation (H) on polyP accumulation. PolyP was extracted from the indicated strains grown in LB or LB + iron (200 μm FeSO4) for 1.5 h or in MOPS for 3 h following a shift from LB and analyzed on TBE-urea gels stained with toluidine blue. Note: the same BasR-tagged strains used for S2F were used for S2G polyP extraction, and the same ArnC-tagged strains used for 3I were used for the polyP extraction shown in S3H. Images shown are representative of results from ≥3 experiments, except for the polyP extractions in S3G and S3H, which are representative of 2 independent replicates. (TIF)</p

Broad proteomic changes in Δ<i>ppk</i> cells during stress.

(A) Experimental set up for proteomics analysis. Cells were grown in LB media to mid-exponential phase before a shift into MOPS minimal media (0.1 mM K2HPO4, 0.4% glucose) for 3 h to induce amino acid starvation and polyP accumulation. The experiment was conducted using n = 5 biological replicates. (B) Volcano plot of significantly differentially expressed proteins (log2(fold-change Δppk/WT)). In red and blue are the significantly up-regulated proteins (FDR-adjusted p-value ppk strains, respectively. (C) Bubble plot showing the “all-or-none” proteins detected only in either wild-type cells or Δppk mutants. Data represent the raw protein spectral counts in 5 biological replicates from each condition. (D) Select confirmations of mass spectrometry data. Chromosomally C-terminal 3Flag-tagged strains were grown under the same conditions used for the mass spectrometry analysis. Protein extracts were resolved using a 12% (for YbdL-3Flag) and 10% (for all other proteins) SDS-PAGE gel, transferred to PVDF membrane, and probed using an anti-Flag antibody. Images are representative of results from ≥3 experiments. (E) GO terms that are significantly enriched among the differentially expressed and “all-or-none proteins” identified by mass spectrometry analysis. (F) GO terms deemed differentially expressed based on GSEA for wild-type and Δppk mutant cells. The underlying data for Fig 1B, 1C, and 1E can be found in S1 Data. FDR, false discovery rate; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis.</p

Raw data for the growth curves and growth curve analysis presented in Fig 2A–2D.

Raw data for the growth curves and growth curve analysis presented in Fig 2A–2D.</p

Wild-type <i>E</i>. <i>coli</i> accumulate polyP in MOPS minimal media while Δ<i>ppk</i> mutants do not.

PolyP extraction gel from wild-type and Δppk mutant cultures used for mass spectrometry sample preparation. Overnight cultures were grown in LB media to mid-exponential phase and then shifted into MOPS minimal media for 3 h to induce starvation and polyP accumulation. PolyP extracts were run on a TBE-urea gel and stained with toluidine blue. The migration of a chain approximately 700 phosphate residues in length (p700) is indicated. (TIF)</p

Raw data for volcano plot, all-or-none protein bubble plot, and GO term analysis presented in Fig 1B, 1C and 1E.

Raw data for volcano plot, all-or-none protein bubble plot, and GO term analysis presented in Fig 1B, 1C and 1E.</p