Raman spectroscopy-based measurements of single-cell phenotypic diversity in microbial populations

Microbial cells experience physiological changes due to environmental change, such as pH and temperature, the release of bactericidal agents, or nutrient limitation. This has been shown to affect community assembly and physiological processes (e.g., stress tolerance, virulence, or cellular metabolic activity). Metabolic stress is typically quantified by measuring community phenotypic properties such as biomass growth, reactive oxygen species, or cell permeability. However, bulk community measurements do not take into account single-cell phenotypic diversity, which is important for a better understanding and the subsequent management of microbial populations. Raman spectroscopy is a nondestructive alternative that provides detailed information on the biochemical makeup of each individual cell. Here, we introduce a method for describing single-cell phenotypic diversity using the Hill diversity framework of Raman spectra. Using the biomolecular profile of individual cells, we obtained a metric to compare cellular states and used it to study stress-induced changes. First, in two Escherichia coli populations either treated with ethanol or nontreated and then in two Saccharomyces cerevisiae subpopulations with either high or low expression of a stress reporter. In both cases, we were able to quantify single-cell phenotypic diversity and to discriminate metabolically stressed cells using a clustering algorithm. We also described how the lipid, protein, and nucleic acid compositions changed after the exposure to the stressor using information from the Raman spectra. Our results show that Raman spectroscopy delivers the necessary resolution to quantify phenotypic diversity within individual cells and that this information can be used to study stress-driven metabolic diversity in microbial populations. IMPORTANCE Microbial cells that live in the same community can exist in different physiological and morphological states that change as a function of spatiotemporal variations in environmental conditions. This phenomenon is commonly known as phenotypic heterogeneity and/or diversity. Measuring this plethora of cellular expressions is needed to better understand and manage microbial processes. However, most tools to study phenotypic diversity only average the behavior of the sampled community. In this work, we present a way to quantify the phenotypic diversity of microbial samples by inferring the (bio)molecular profile of its constituent cells using Raman spectroscopy. We demonstrate how this tool can be used to quantify the phenotypic diversity that arises after the exposure of microbes to stress. Raman spectroscopy holds potential for the detection of stressed cells in bioproduction

Detecting phenotypes with Raman spectroscopy

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Unwanted coliforms can hide in mature drinking water biofilms, grown in full-scale distribution networks

AbstractBiofilms within drinking water distribution systems are an important habitat for drinking water microorganisms. However, they can play a role in microbial regrowth, water discoloration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy and 16S rRNA sequencing. Overall, biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. We observed that water sources shaped the biofilm community composition while drinking water disinfection determined the biofilm density. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform,Serratia fonticola. After spiking low concentrations (i.e. ± 100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes are emphasizing the need for continued research on biofilm detachment and its implications for water contamination in distribution networks.</jats:p

The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation

HIV-1-mediated CD4 down-regulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-Binding protein (HRB) compared to control cells. HRB knock-down affected CD4 down-regulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB Like), but not of the HRB interaction partner EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both these proteins are known to be involved in HIV-1-mediated CD4 down-regulation as cofactors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 down-regulation and highlight differences with the related protein HRBL, which affects the CD4 down-regulation in a dual role as co-factor of both HIV-1 Nef and Vpu