In situ single-cell activities of microbial populations revealed by spatial transcriptomics

D, Dar; N, Dar; Cai, Liang; Dk, Newman

doi:10.1101/2021.02.24.432792

Cited by 11 publications

(8 citation statements)

References 100 publications

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“…strains ( Table 1 ), which corroborates the performance of this probe, providing a suitable alternative for studies in pure cultures but also in biofilms. The main advantage of using PNA rRNA probes is related with the visualization of the whole cells and spatial distribution and metabolic activity of the cells [ 45 ], allowing a better knowledge of the organization and functional development of biofilms.…”

Section: Discussionmentioning

confidence: 99%

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

et al. 2022

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Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of Legionella.

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Section: Discussionmentioning

confidence: 99%

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

et al. 2022

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“…Moreover, recent developments combining barcoding with fluorescence in situ hybridization have increased the spatial resolution at which cell lineage can be traced [ 135 ] and dual-view light sheet microscopy has made it possible to track entire cell populations in real time without interruption [ 127 , 136 ]. Finally, advances in spatial transcriptomics make it possible to simultaneously examine the expression of hundreds of genes in space, which, for instance, revealed that bacteria cells can express vastly distinct expression profiles within the same colony [ 137 ]. Currently, most of the above technologies are applied to study a few genetically tractable model organisms, but they hold the promise of uncovering many still hidden forms of multicellular organization in nonmodel organisms as well.…”

Section: Discussionmentioning

confidence: 99%

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation

Gestel

Wagner

2021

PLoS Biol

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The repeated evolution of multicellularity leads to a wide diversity of organisms, many of which are sessile, including land plants, many fungi, and colonial animals. Sessile organisms adhere to a surface for most of their lives, where they grow and compete for space. Despite the prevalence of surface-associated multicellularity, little is known about its evolutionary origin. Here, we introduce a novel theoretical approach, based on spatial lineage tracking of cells, to study this origin. We show that multicellularity can rapidly evolve from 2 widespread cellular properties: cell adhesion and the regulatory control of adhesion. By evolving adhesion, cells attach to a surface, where they spontaneously give rise to primitive cell collectives that differ in size, life span, and mode of propagation. Selection in favor of large collectives increases the fraction of adhesive cells until a surface becomes fully occupied. Through kin recognition, collectives then evolve a central-peripheral polarity in cell adhesion that supports a division of labor between cells and profoundly impacts growth. Despite this spatial organization, nascent collectives remain cryptic, lack well-defined boundaries, and would require experimental lineage tracking technologies for their identification. Our results suggest that cryptic multicellularity could readily evolve and originate well before multicellular individuals become morphologically evident.

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“…In addition to these single-cell technologies, earlier this year the Newman and Cai laboratories at Caltech successfully generated spatial single-cell bacterial transcriptomics in Pseudomonas aeruginosa using a microscopical approach. This dataset probed a large number of cells (∼600,000) but limited the transcript coverage to 105 genes ( Dar et al, 2021 ) as opposed to studies done at the full transcriptome scale. In order to identify distinct cellular states in bacteria with inherently low abundance of transcripts, capturing the highest number of transcripts and number of cells are the two most critical parameters.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell RNA Sequencing Elucidates the Structure and Organization of Microbial Communities

Brennan¹,

Rosenthal²

2021

Front. Microbiol.

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Clonal bacterial populations exhibit various forms of heterogeneity, including co-occurrence of cells with different morphological traits, biochemical properties, and gene expression profiles. This heterogeneity is prevalent in a variety of environments. For example, the productivity of large-scale industrial fermentations and virulence of infectious diseases are shaped by cell population heterogeneity and have a direct impact on human life. Due to the need and importance to better understand this heterogeneity, multiple methods of examining single-cell heterogeneity have been developed. Traditionally, fluorescent reporters or probes are used to examine a specific gene of interest, providing a useful but inherently biased approach. In contrast, single-cell RNA sequencing (scRNA-seq) is an agnostic approach to examine heterogeneity and has been successfully applied to eukaryotic cells. Unfortunately, current extensively utilized methods of eukaryotic scRNA-seq present difficulties when applied to bacteria. Specifically, bacteria have a cell wall which makes eukaryotic lysis methods incompatible, bacterial mRNA has a shorter half-life and lower copy numbers, and isolating an individual bacterial species from a mixed community is difficult. Recent work has demonstrated that these technical hurdles can be overcome, providing valuable insight into factors influencing microbial heterogeneity. This perspective describes the emerging microbial scRNA-seq toolkit. We outline the benefit of these new tools in elucidating numerous scientific questions in microbiological studies and offer insight about the possible rules that govern the segregation of traits in individual microbial cells.

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In situ single-cell activities of microbial populations revealed by spatial transcriptomics

Cited by 11 publications

References 100 publications

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation

Single-Cell RNA Sequencing Elucidates the Structure and Organization of Microbial Communities

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