Editor's Pick Research Article

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

Rishi R. Trivedi, John A. Crooks, George K. Auer, Joel Pendry, Ilona P. Foik, Albert Siryaporn, Nicholas L. Abbott, Zemer Gitai, Douglas B. Weibel
Vernita Gordon, Invited Editor, Marvin Whiteley, Editor
DOI: 10.1128/mBio.01340-18

Article Figures & Data

Figures

  • FIG 1 
    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 
    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 
    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 
    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 
    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 
    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.

Supplemental Material

Back to top
Download PDF
Citation Tools
Print

Alerts
Email

KEYWORDS

cell stiffness
DadA
mechanical genomics
Pseudomonas aeruginosa
cell wall

Related Articles

Cited By...