Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness

Abstract

The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as Pseudomonas aeruginosa that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated P. aeruginosa individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for P. aeruginosa d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes ponA and dacC Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from P. aeruginosa cells revealed that dadA deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.IMPORTANCE The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen Pseudomonas aeruginosa This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of P. aeruginosa cells. We demonstrate that d-Ala-a component of the peptidoglycan-is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential P. aeruginosa targets that may enhance antibacterial chemotherapies or lead to new approaches.

Keywords: DadA; Pseudomonas aeruginosa; cell stiffness; cell wall; mechanical genomics.

FIG 1

Biochemistry of d-Ala in Gram-negative bacteria. The cartoon represents the utilization and role of d-Ala in bacterial cells. P. aeruginosa cells have two alanine racemases (Alr and DadX) that interconvert l-Ala and d-Ala. DadA is a d-amino-acid dehydrogenase that degrades d-Ala into pyruvate. Ddl is an amino acid ligase that converts two d-Ala molecules into d-Ala-d-Ala, which is a substrate of the enzyme MurF in forming lipid I from the MurNAc tripeptide. MraY and MurG form lipid II, which is subsequently flipped across the membrane into the periplasm and incorporated into the growing peptidoglycan. The PonA transpeptidase cross-links stem peptides during peptidoglycan biosynthesis by releasing the terminal d-Ala into the periplasm. dd-Carboxypeptidase (DacC) and dd-endopeptidases (PbpG) also release the terminal d-Ala from the un-cross-linked lipid II in the periplasm. Free d-Ala in the periplasm and in the extracellular environment is transported into cells through alanine transporters and permeases. “PP-lipid” refers to a diphosphate bridge and long, connected hydrocarbon tail that is attached to the disaccharide in lipid II.

FIG 2

Genome-wide stiffness screen of Pseudomonas aeruginosa. (A) A scatter plot of all gene transposon mutants in P. aeruginosa PA14 and corresponding absorbance values (λ = 595 nm) for cell growth in LB and in 1% agarose infused with LB; each point represents a single gene transposon mutant. Regions in the plot with the highest density of data points are depicted in yellow. (B) A plot of genome-wide stiffness screening data fitted using a bivariate normal distribution. Transposon mutants highlighted by blue data points (n = 115 genes) had higher growth in 1% agarose than the mutants highlighted by red data points (n = 133 genes). Genes that lie within the interval between two dashed lines (in gray) followed a linear growth model. (C) A summary of KEGG pathway enrichment for the data depicted in blue (in panel B) with higher cell stiffness and the data depicted in red with lower cell stiffness.

FIG 3

Stiffness genes code proteins involved in diverse biochemical pathways. (A) A histogram depicting that the GRABS score distribution of rescreened genes (bottom panel) shows a reduction in the mean GRABS score compared to the genes in the entire screen (top panel). (B) A plot of the GRABS scores along with the corresponding variance for 42 rescreened genes. 36 out of 42 genes were annotated and had an assigned biochemical function. 6 of the top 42 hits are not yet annotated and are named after their respective gene locus. The P. aeruginosa PA14 dadA::Tn mutant (depicted in a red) has very low variance in the GRABS score and consistently produces a negative GRABS score. (C) Gene ontology information (COG, classification of gene ontology) for the top hits. The numbers surrounding the pie chart indicate the number of genes (out of the 42 selected) that are represented within each COGs family.

FIG 4

d-Ala dehydrogenase (DadA) is a modulator of P. aeruginosa cell stiffness. (Left panel) GRABS score for P. aeruginosa wild-type cells, dadA::Tn cells, and the dadA complementation strain. (Right panel) Young’s modulus of wild-type cells, dadA::Tn cells, and the dadA complementation strain. Stiffness measurements were performed using a microfluidic cell-bending assay.

FIG 5

The peptidoglycan biosynthetic pathway is sensitive to d-Ala levels. (A) P. aeruginosa PA14 dadA::Tn mutant cells grew better than the PA14 wild-type strain in the presence of a sub-MIC level of DCS. y-axis data represents absorbance (λ = 595 nm) after 16 h of growth. Adding d-Ala to the growth media enhanced the growth phenotype of the dadA::Tn mutant. (B) An increase in the concentration of exogenous d-Ala (in the growth media) reduced the GRABS score for dadA::Tn mutant cells compared to wild-type cells. (C) dadA::Tn mutant cells grown in LB media supplemented with d-Ala-d-Ala (15 mM) had a 30% decrease in the GRABS score compared to wild-type cells. (D) Transcription of ponA, dacC, murF, and ddl was reduced in P. aeruginosa PA14 dadA::Tn mutant cells compared to wild-type cells. These genes code for proteins that either release d-Ala into the periplasm (dacC and ponA) or utilize d-Ala as a substrate for peptidoglycan precursor synthesis (ddl and murF).

FIG 6

The peptidoglycan composition of dadA::Tn mutant cells is altered compared to P. aeruginosa wild-type cells. (A) UPLC-MS data revealed that the muropeptide composition of the P. aeruginosa wild-type strain and that of the dadA mutant differ in the abundance of monomer, dimer, and anhydrously terminated saccharides. n = 3 biological replicates. (B) We observed a decrease in peptidoglycan cross-linking of dadA::Tn compared to wild-type cells. Error bars represent standard deviations of the means.