CaltechAUTHORS
  A Caltech Library Service

Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

Jorth, Peter and Spero, Melanie A. and Livingston, J. and Newman, Dianne K. (2019) Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone. mBio, 10 (6). Art. No. e02622-19. ISSN 2150-7511. PMCID PMC6918079. https://resolver.caltech.edu/CaltechAUTHORS:20190510-105529016

[img] PDF - Published Version
Creative Commons Attribution.

1763Kb
[img] PDF - Submitted Version
Creative Commons Attribution No Derivatives.

489Kb
[img] Image (TIFF) (FIG S1 Individual P. aeruginosa HCR v3.0 probes are specific) - Supplemental Material
Creative Commons Attribution.

2738Kb
[img] Image (TIFF) (FIG S2 Initiators probes do not induce amplification when only one probe in the initiator pair is used. HCR v3.0 initiator probes come in pairs of odd and even initiators) - Supplemental Material
Creative Commons Attribution.

1779Kb
[img] Image (TIFF) (FIG S3 Two-channel algD HCR analysis verifies that new P. aeruginosa algD HCR probes are quantitative) - Supplemental Material
Creative Commons Attribution.

1254Kb
[img] Image (TIFF) (FIG S4 ABBA nonmucoid and mucoid aggregates grow faster and are larger near the air-agar interface) - Supplemental Material
Creative Commons Attribution.

527Kb
[img] Image (TIFF) (FIG S5 Alginate and nitrate reductase gene expression is generally higher in mucoid P. aeruginosa than in nonmucoid P. aeruginosa in 2 ABBA experiments) - Supplemental Material
Creative Commons Attribution.

1694Kb
[img] Image (TIFF) (FIG S6 Alginate gene expression is induced most highly in planktonic cultures by anoxia) - Supplemental Material
Creative Commons Attribution.

82Kb
[img] MS Word (TABLE S1 HCR probe target sequences) - Supplemental Material
Creative Commons Attribution.

22Kb
[img] MS Word (TABLE S2 Bacterial strains) - Supplemental Material
Creative Commons Attribution.

20Kb
[img] MS Word (TABLE S3 Primers) - Supplemental Material
Creative Commons Attribution.

20Kb
[img] MS Word (TEXT S1 Supplemental methods) - Supplemental Material
Creative Commons Attribution.

31Kb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20190510-105529016

Abstract

It is well appreciated that oxygen- and other 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 provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 μM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections. IMPORTANCE: A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1128/mbio.02622-19DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918079PubMed CentralArticle
https://doi.org/10.1101/632893DOIDiscussion Paper
ORCID:
AuthorORCID
Jorth, Peter0000-0002-0981-740X
Spero, Melanie A.0000-0003-3291-2138
Newman, Dianne K.0000-0003-1647-1918
Alternate Title:Quantitative visualization of gene expression in Pseudomonas aeruginosa aggregates reveals peak expression of alginate in the hypoxic zone
Additional Information:© 2019 Jorth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Received 3 October 2019. Accepted 1 November 2019. Published 17 December 2019. We thank Will DePas, Ruth Lee, Niles Pierce, Maayan Schwarzkopf, 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 D.K.N. from the Army Research Office (W911NF-17-1-0024) and National Institutes of Health (1R01AI127850-01A1 and 1R21AI146987-01) supported this research. P.J. was supported by postdoctoral fellowships from the Cystic Fibrosis Foundation (JORTH14F0 and JORTH17F5) and a grant from the National Institutes of Health (1K22AI127473-01A1). M.A.S. 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
NIH1R21AI146987-01
Cystic Fibrosis FoundationJORTH14F0
Cystic Fibrosis FoundationJORTH17F5
NIH1K22AI127473-01A1
Doren Family FoundationUNSPECIFIED
Subject Keywords:HCR, Pseudomonas aeruginosa, aggregate, biofilms, gene expression, in situ hybridization, microscopy
Issue or Number:6
PubMed Central ID:PMC6918079
Record Number:CaltechAUTHORS:20190510-105529016
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190510-105529016
Official Citation:Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone Peter Jorth, Melanie A. Spero, J. Livingston, Dianne K. Newman mBio Dec 2019, 10 (6) e02622-19; DOI: 10.1128/mBio.02622-19
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:12 Feb 2020 18:28

Repository Staff Only: item control page