Direct Observation of the Dynamics of Single-Cell Metabolic Activity during Microbial Diauxic Growth

Abstract

Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology’s most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph Methylobacterium extorquens. In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches. IMPORTANCE Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.

The oral microbiome: Role of key organisms and complex networks in oral health and disease

“…Single‐cell sequencing was developed initially for immune‐profiling purposes but has been adapted to permit assessments of single microbial cells 91 . Assessing individual cells from environmental samples increases the detection rate of unculturable organisms while providing the opportunity to ask more novel questions related to the functional capacities and significant roles of single organisms within complex microbial communities 92,93 . Application of transcriptomic approaches has proved extremely significant for delineating both microbial and host gene expression in the context of oral health and pathology 14,68,94 …”

“…91 Assessing individual cells from environmental samples increases the detection rate of unculturable organisms while providing the opportunity to ask more novel questions related to the functional capacities and significant roles of single organisms within complex microbial communities. 92,93 Application of transcriptomic approaches has proved extremely significant for delineating both microbial and host gene expression in the context of oral health and pathology. 14,68,94 The field faces some key hurdles, namely the challenge of separating out the highly complex mixtures that are typical of clinical samples while simultaneously visualizing many molecules of a diverse chemical nature.…”

Comparative Genomic Analysis of a Methylorubrum rhodesianum MB200 Isolated from Biogas Digesters Provided New Insights into the Carbon Metabolism of Methylotrophic Bacteria

“…MM contains three parts: the phosphate salts solution [(NH 4 ) 2 HPO 4 3 g/L, K 2 HPO 4 2 g/L, and NaCl 1 g/L in 1000 mL distilled water; pH 7.0), solution A (MgSO 4 ·7H 2 O 20 g/L, CaCl 2 ·2H 2 O 2 g/L, and FeSO 4 2 g/L in 100 mL distilled water), and solution B (MnSO 4 ·7H 2 O 0.5 g/L, Na 2 MoO 4 ·2H 2 O 0.5 g/L, and ZnSO 4 ·7H 2 O 0.5 g/L in 100 mL distilled water). Trace biotin was added at a ratio of 1/10,000 in the medium [ 50 ]. The following concentrations of each carbon source were added to MM medium before use: formaldehyde (0.21, 0.42, and 0.84 mM; “FM”), methanol (125, 250, and 375 mM; “MA”), ethanol (86, 172, and 258 mM; “EA”), and succinate (102,153, and 204 mM; “SA”).…”

Single-cell Raman spectroscopy identifies Escherichia coli persisters and reveals their enhanced metabolic activities

“…Traditionally, measurement of metabolic levels focuses on a single pathway like respiratory activity (Huang et al, 2019 ), or complicated metabonomic–proteomic techniques are performed to evaluate the total metabolites of the whole microbial population (Li et al, 2018 ). It is worthy to note that newly developed approaches enable us to track the fate of essential chemical elements, such as hydrogen, carbon, and nitrogen, using their isotopes (Wang et al, 2016 ; Nikolic et al, 2017 ; McClelland et al, 2020 ). As H 2 O is an essential element of metabolism in living cells, tracking and quantifying the transition from H to D are excellent alternatives to assess the metabolic activities of the targeting microbes and their persisters (Tao et al, 2017 ).…”