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

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.

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Quantitative visualization of gene expression inPseudomonas aeruginosaaggregates reveals peak expression of alginate in the hypoxic zone

10.1101/632893 ◽

2019 ◽

Cited By ~ 2

Author(s):

Peter Jorth ◽

Melanie A. Spero ◽

Dianne K. Newman

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Spatial Scales ◽

Hybridization Chain Reaction ◽

Microbial Activities ◽

Chronic Infections ◽

Chain Reaction ◽

Agar Block ◽

Bacterial Gene

AbstractIt 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 scalesin situ. Here we report the application of the hybridization chain reaction (HCR) v3.0 toPseudomonas aeruginosaaggregates 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 quantifyalgDandnarGgene 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 μM 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.ImportanceThe visualization of microbial activities in natural environments is an important goal for numerous studies in microbial ecology, be the environment a sediment, soil, or infected human tissue. Here we report the application of the hybridization chain reaction (HCR) v3.0 to measure microbial gene expressionin situat single-cell resolution in aggregate biofilms. UsingPseudomonas aeruginosawith a tunable gene expression system, we show that this methodology is quantitative. Leveraging HCR v3.0 to measure gene expression within aP. aeruginosaaggregate, we find 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, HCR v3.0 has potential for broad application into microbial activitiesin situat small spatial scales.

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Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

10.1101/2021.08.02.454831 ◽

2021 ◽

Author(s):

Julia C Duckhorn ◽

Ian P Junker ◽

Yun Ding ◽

Troy R Shirangi

Keyword(s):

Gene Expression ◽

Nervous System ◽

In Situ Hybridization ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Nervous Systems ◽

Whole Mount ◽

Central Nervous Systems ◽

Mrna And Protein Expression

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.

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Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

10.1101/214619 ◽

2017 ◽

Author(s):

Vikas Trivedi ◽

Harry M.T. Choi ◽

Scott E. Fraser ◽

Niles A. Pierce

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Mrna Expression ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Developmental Gene Expression ◽

Vertebrate Embryos ◽

Anatomical Space ◽

Subcellular Resolution

ABSTRACTFor decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting, and/or homogenization. Here, we eliminate the tradeoff between quantitation and anatomical context, using multiplexed in situ hybridization chain reaction (HCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable iterative bi-directional queries that open a new era for in situ hybridization.SUMMARYMultiplexed in situ hybridization chain reaction (HCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.

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Single-Cell Analysis Reveals that the Enterococcal Sex Pheromone Response Results in Expression of Full-Length Conjugation Operon Transcripts in All Induced Cells

Journal of Bacteriology ◽

10.1128/jb.00685-19 ◽

2020 ◽

Vol 202 (8) ◽

Author(s):

Rebecca J. B. Erickson ◽

Arpan A. Bandyopadhyay ◽

Aaron M. T. Barnes ◽

Sofie A. O’Brien ◽

Wei-Shou Hu ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Antibiotic Treatment ◽

Single Cell Analysis ◽

Single Cells ◽

Group Behavior ◽

Early Gene ◽

Hybridization Chain Reaction ◽

Cell Analysis ◽

Content Type

ABSTRACT For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an “early” gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (QL, prgB, and prgA) also expressed late transcripts (prgJ, pcfC, and pcfG). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells. IMPORTANCE In Enterococcus faecalis, conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer.

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Use of Hybridization Chain Reaction-FluorescentIn SituHybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis

Applied and Environmental Microbiology ◽

10.1128/aem.00890-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4728-4735 ◽

Cited By ~ 24

Author(s):

K. Nikolakakis ◽

E. Lehnert ◽

M. J. McFall-Ngai ◽

E. G. Ruby

Keyword(s):

Gene Expression ◽

Vibrio Fischeri ◽

Hybridization Chain Reaction ◽

Bacterial Cells ◽

Chain Reaction ◽

Content Type ◽

Symbiotic Associations ◽

Spatiotemporal Regulation ◽

Habitat Transition ◽

Regulated Gene Expression

ABSTRACTThe establishment of a productive symbiosis betweenEuprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont,Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescentin situhybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in bothE. scolopesandV. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization.

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