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Quantitative visualization of gene expression in Pseudomonas aeruginosa aggregates reveals peak expression of alginate in the hypoxic zone

Jorth, Peter and Spero, Melanie A. and Newman, Dianne K. (2019) Quantitative visualization of gene expression in Pseudomonas aeruginosa aggregates reveals peak expression of alginate in the hypoxic zone. . (Unpublished) http://resolver.caltech.edu/CaltechAUTHORS:20190510-105529016

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Abstract

It is well appreciated that oxygen- and nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ. Here we report the application of the hybridization chain reaction (HCR) v3.0 to Pseudomonas aeruginosa aggregates as a step towards this end. As proof-of-principle, we visualize the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG). Using an inducible bacterial gene expression construct to calibrate the HCR signal, we were able to quantify algD and narG gene expression across microenvironmental gradients both within single aggregates and within aggregate populations using the Agar Block Biofilm Assay (ABBA). For the ABBA population, alginate gene expression was restricted to hypoxic regions within the environment (~40-200 mM O2), as measured by an oxygen microelectrode. Within individual biofilm aggregates, cells proximal to the surface expressed alginate genes to a greater extent than interior cells. Lastly, mucoid biofilms consumed more oxygen than nonmucoid biofilms. These results establish that HCR has a sensitive dynamic range and can be used to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. Because HCR v3.0 can be performed on diverse cell types, this methodological advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen behavior in human chronic infections.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/632893DOIDiscussion Paper
ORCID:
AuthorORCID
Jorth, Peter0000-0002-0981-740X
Newman, Dianne K.0000-0003-1647-1918
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license. We would like to thank Will DePas, Ruth Lee, Niles Pierce and the Programmable Molecular Technology Center at the Caltech Beckman Institute for technical assistance and advice. 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. Grants to DKN from the Army Research Office (W911NF-17-1-0024) and National Institutes of Health (1R01AI127850-01A1) supported this research. PJ was supported by postdoctoral fellowships from the Cystic Fibrosis Foundation (JORTH14F0 and JORTH17F5). MAS was supported by a gift from the Doren Family Foundation.
Funders:
Funding AgencyGrant Number
Arnold and Mabel Beckman FoundationUNSPECIFIED
Army Research Office (ARO)W911NF-17-1-0024
NIH1R01AI127850-01A1
Cystic Fibrosis FoundationJORTH14F0
Cystic Fibrosis FoundationJORTH17F5
Doren Family FoundationUNSPECIFIED
Record Number:CaltechAUTHORS:20190510-105529016
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20190510-105529016
Official Citation:Quantitative visualization of gene expression in Pseudomonas aeruginosa aggregates reveals peak expression of alginate in the hypoxic zone Peter Jorth, Melanie Spero, Dianne K. Newman bioRxiv 632893; doi: https://doi.org/10.1101/632893
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95390
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:10 May 2019 20:24
Last Modified:10 May 2019 20:24

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