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Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism

Livingston, Jadzia and Spero, Melanie A. and Lonergan, Zachery R. and Newman, Dianne K. (2022) Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism. Applied and Environmental Microbiology, 88 (11). Art. No. aem.00439-22. ISSN 0099-2240. doi:10.1128/aem.00439-22.

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Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations.

Item Type:Article
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URLURL TypeDescription Material Paper
Spero, Melanie A.0000-0003-3291-2138
Newman, Dianne K.0000-0003-1647-1918
Additional Information:© 2022 American Society for Microbiology. Received 10 March 2022; Accepted 28 April 2022; Published 19 May 2022. We thank Lars Dietrich and Lindsey Christine Florek for the ΔccoN1 PA14 strain, Steven Wilbert for the ΔnirS and ΔnosZ strains, and David Basta and Nate Glasser for the ΔackA strain. We thank Chelsey VanDrisse, Steven Wilbert, John Ciemnieki, and Georgia Squyres for constructive feedback, and we thank Niles Pierce and Maayan Schwarzkopf for assistance using the HCR system. Confocal microscopy was performed in the Caltech Biological Imaging Facility at the Caltech Beckman Institute, which is supported by the Arnold and Mabel Beckman Foundation, and we thank Giada Spigolon for her help using the facility. This work was supported by grants from the NIH (R01 HL152190-03) to D.K.N., the Doren Family Foundation, the Cystic Fibrosis Foundation (postdoctoral fellowship to M.A.S., SPERO19F0), and the Jane Coffin Childs Memorial Fund (postdoctoral fellowship to Z.R.L.).
Funding AgencyGrant Number
Arnold and Mabel Beckman FoundationUNSPECIFIED
NIHR01 HL152190-03
Doren Family FoundationUNSPECIFIED
Cystic Fibrosis FoundationSPERO19F0
Jane Coffin Childs Memorial Fund for Medical ResearchUNSPECIFIED
Subject Keywords:ABBA, HCR, Pseudomonas aeruginosa, aggregates, biofilms, denitrification, gene expression, heterogeneity, hybridization chain reaction
Issue or Number:11
Record Number:CaltechAUTHORS:20220315-626233000
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:113920
Deposited By: George Porter
Deposited On:16 Mar 2022 17:04
Last Modified:29 Jun 2022 13:53

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