Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> 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 bacte...

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Autores principales:	Rishi R. Trivedi, John A. Crooks, George K. Auer, Joel Pendry, Ilona P. Foik, Albert Siryaporn, Nicholas L. Abbott, Zemer Gitai, Douglas B. Weibel
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	cell stiffness DadA mechanical genomics Pseudomonas aeruginosa cell wall Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b0e1f27012c8493db4b737bc9e32c109
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spelling	oai:doaj.org-article:b0e1f27012c8493db4b737bc9e32c1092021-11-15T15:58:21ZMechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness10.1128/mBio.01340-182150-7511https://doaj.org/article/b0e1f27012c8493db4b737bc9e32c1092018-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01340-18https://doaj.org/toc/2150-7511ABSTRACT 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.Rishi R. TrivediJohn A. CrooksGeorge K. AuerJoel PendryIlona P. FoikAlbert SiryapornNicholas L. AbbottZemer GitaiDouglas B. WeibelAmerican Society for Microbiologyarticlecell stiffnessDadAmechanical genomicsPseudomonas aeruginosacell wallMicrobiologyQR1-502ENmBio, Vol 9, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell stiffness DadA mechanical genomics Pseudomonas aeruginosa cell wall Microbiology QR1-502
spellingShingle	cell stiffness DadA mechanical genomics Pseudomonas aeruginosa cell wall Microbiology QR1-502 Rishi R. Trivedi John A. Crooks George K. Auer Joel Pendry Ilona P. Foik Albert Siryaporn Nicholas L. Abbott Zemer Gitai Douglas B. Weibel Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
description	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.
format	article
author	Rishi R. Trivedi John A. Crooks George K. Auer Joel Pendry Ilona P. Foik Albert Siryaporn Nicholas L. Abbott Zemer Gitai Douglas B. Weibel
author_facet	Rishi R. Trivedi John A. Crooks George K. Auer Joel Pendry Ilona P. Foik Albert Siryaporn Nicholas L. Abbott Zemer Gitai Douglas B. Weibel
author_sort	Rishi R. Trivedi
title	Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
title_short	Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
title_full	Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
title_fullStr	Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
title_full_unstemmed	Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness
title_sort	mechanical genomic studies reveal the role of <sc>d</sc>-alanine metabolism in <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> cell stiffness
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/b0e1f27012c8493db4b737bc9e32c109
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Mechanical Genomic Studies Reveal the Role of <sc>d</sc>-Alanine Metabolism in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Cell Stiffness

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