Bacterial phenotypic heterogeneity through the lens of single-cell RNA sequencing.

Bacterial transcription is not monolithic. Microbes exist in a wide variety of cell states that help them adapt to their environment, acquire and produce essential nutrients, and engage in both competition and cooperation with their neighbors. While we typically think of bacterial adaptation as a group behavior, where all cells respond in unison, there is often a mixture of phenotypic responses within a bacterial population, where distinct cell types arise. A primary phenomenon driving these distinct cell states is transcriptional heterogeneity. Given that bacterial mRNA transcripts are extremely short-lived compared to eukaryotes, their transcriptional state is closely associated with their physiology, and thus the transcriptome of a bacterial cell acts as a snapshot of the behavior of that bacterium. Therefore, the application of single-cell transcriptomics to microbial populations will provide novel insight into cellular differentiation and bacterial ecology. In this review, we provide an overview of transcriptional heterogeneity in microbial systems, discuss the findings already provided by single-cell approaches, and plot new avenues of inquiry in transcriptional regulation, cellular biology, and mechanisms of heterogeneity that are made possible when microbial communities are analyzed at single-cell resolution.

Probe-based bacterial single-cell RNA sequencing predicts toxin regulation.

Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.

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

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.

Murine single-cell RNA-seq reveals cell-identity- and tissue-specific trajectories of aging.

Aging is a pleiotropic process affecting many aspects of mammalian physiology. Mammals are composed of distinct cell type identities and tissue environments, but the influence of these cell identities and environments on the trajectory of aging in individual cells remains unclear. Here, we performed single-cell RNA-seq on >50,000 individual cells across three tissues in young and old mice to allow for direct comparison of aging phenotypes across cell types. We found transcriptional features of aging common across many cell types, as well as features of aging unique to each type. Leveraging matrix factorization and optimal transport methods, we found that both cell identities and tissue environments exert influence on the trajectory and magnitude of aging, with cell identity influence predominating. These results suggest that aging manifests with unique directionality and magnitude across the diverse cell identities in mammals.

Metabolic interactions between dynamic bacterial subpopulations.

Individual microbial species are known to occupy distinct metabolic niches within multi-species communities. However, it has remained largely unclear whether metabolic specialization can similarly occur within a clonal bacterial population. More specifically, it is not clear what functions such specialization could provide and how specialization could be coordinated dynamically. Here, we show that exponentially growing Bacillus subtilis cultures divide into distinct interacting metabolic subpopulations, including one population that produces acetate, and another population that differentially expresses metabolic genes for the production of acetoin, a pH-neutral storage molecule. These subpopulations exhibit distinct growth rates and dynamic interconversion between states. Furthermore, acetate concentration influences the relative sizes of the different subpopulations. These results show that clonal populations can use metabolic specialization to control the environment through a process of dynamic, environmentally-sensitive state-switching.

Mapping a multiplexed zoo of mRNA expression.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Directed evolution of a far-red fluorescent rhodopsin.

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK(a) of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼ 620 nm/730 nm).

Genome-wide effects of selenium and translational uncoupling on transcription in the termite gut symbiont Treponema primitia.

UNLABELLED: When prokaryotic cells acquire mutations, encounter translation-inhibiting substances, or experience adverse environmental conditions that limit their ability to synthesize proteins, transcription can become uncoupled from translation. Such uncoupling is known to suppress transcription of protein-encoding genes in bacteria. Here we show that the trace element selenium controls transcription of the gene for the selenocysteine-utilizing enzyme formate dehydrogenase (fdhFSec) through a translation-coupled mechanism in the termite gut symbiont Treponema primitia, a member of the bacterial phylum Spirochaetes. We also evaluated changes in genome-wide transcriptional patterns caused by selenium limitation and by generally uncoupling translation from transcription via antibiotic-mediated inhibition of protein synthesis. We observed that inhibiting protein synthesis in T. primitia influences transcriptional patterns in unexpected ways. In addition to suppressing transcription of certain genes, the expected consequence of inhibiting protein synthesis, we found numerous examples in which transcription of genes and operons is truncated far downstream from putative promoters, is unchanged, or is even stimulated overall. These results indicate that gene regulation in bacteria allows for specific post-initiation transcriptional responses during periods of limited protein synthesis, which may depend both on translational coupling and on unclassified intrinsic elements of protein-encoding genes. IMPORTANCE: A large body of literature demonstrates that the coupling of transcription and translation is a general and essential method by which bacteria regulate gene expression levels. However, the potential role of noncanonical amino acids in regulating transcriptional output via translational control remains, for the most part, undefined. Furthermore, the genome-wide transcriptional state in response to translational decoupling is not well quantified. The results presented here suggest that the noncanonical amino acid selenocysteine is able to tune transcription of an important metabolic gene via translational coupling. Furthermore, a genome-wide analysis reveals that transcriptional decoupling produces a wide-ranging effect and that this effect is not uniform. These results exemplify how growth conditions that impact translational processivity can rapidly feed back on transcriptional productivity of prespecified groups of genes, providing bacteria with an efficient response to environmental changes.

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

Following evolution of bacterial antibiotic resistance in real time.

A new study reports the development of the 'morbidostat', a device that allows for continuous culture of bacteria under a constant drug selection pressure using computer feedback control of antibiotic concentration. This device, together with bacterial whole-genome sequencing, allowed the authors to follow the evolution of resistance-conferring mutations in Escherichia coli populations in real time, providing support for deterministic evolution of resistance in some situations.

RNA-seq reveals cooperative metabolic interactions between two termite-gut spirochete species in co-culture.

The hindguts of wood-feeding termites typically contain hundreds of microbial species. Together with their insect host, these gut microbes degrade lignocellulose into usable catabolites. Although past research revealed many facets of the stepwise flow of metabolites in this scheme, not much is known about the breadth of interactions occurring between termite-gut microbes. Most of these microbes are thought to depend on, and to have co-speciated with, their host and each other for millions of years. In this study, we explored the interactions of two spirochetes previously isolated from the very same termite species. As hydrogen (H(2)) is the central free intermediate in termite-gut lignocellulose digestion, we focused on interactions between two closely related termite-gut spirochetes possessing complementary H(2) physiologies: one produces H(2), while the other consumes it. In vitro, these two Treponema species markedly enhanced each other's growth. RNA sequencing resolved the transcriptomes of these two closely related organisms, revealing that co-cultivation causes comprehensive changes in global gene expression. The expression of well over a 100 genes in each species was changed >twofold, with over a dozen changed >10-fold. Several changes implicating synergistic cross-feeding of known metabolites were validated in vitro. Additionally, certain activities beneficial to the host were preferentially expressed during consortial growth. However, the majority of changes in gene expression are not yet understandable, but indicate a broad, comprehensive and mutualistic interaction between these closely related, co-resident gut symbionts. The results suggest that staggeringly intricate networks of metabolic and gene interactions drive lignocellulose degradation and co-evolution of termite gut microbiota.

Osmotic Stress.

Escherichia coli and Salmonella encounter osmotic pressure variations in natural environments that include host tissues, food, soil, and water. Osmotic stress causes water to flow into or out of cells, changing their structure, physics, and chemistry in ways that perturb cell functions. E. coli and Salmonella limit osmotically induced water fluxes by accumulating and releasing electrolytes and small organic solutes, some denoted compatible solutes because they accumulate to high levels without disturbing cell functions. Osmotic upshifts inhibit membrane-based energy transduction and macromolecule synthesis while activating existing osmoregulatory systems and specifically inducing osmoregulatory genes. The osmoregulatory response depends on the availability of osmoprotectants (exogenous organic compounds that can be taken up to become compatible solutes). Without osmoprotectants, K+ accumulates with counterion glutamate, and compatible solute trehalose is synthesized. Available osmoprotectants are taken up via transporters ProP, ProU, BetT, and BetU. The resulting compatible solute accumulation attenuates the K+ glutamate response and more effectively restores cell hydration and growth. Osmotic downshifts abruptly increase turgor pressure and strain the cytoplasmic membrane. Mechanosensitive channels like MscS and MscL open to allow nonspecific solute efflux and forestall cell lysis. Research frontiers include (i) the osmoadaptive remodeling of cell structure, (ii) the mechanisms by which osmotic stress alters gene expression, (iii) the mechanisms by which transporters and channels detect and respond to osmotic pressure changes, (iv) the coordination of osmoregulatory programs and selection of available osmoprotectants, and (v) the roles played by osmoregulatory mechanisms as E. coli and Salmonella survive or thrive in their natural environments.

General stress response signalling: unwrapping transcription complexes by DNA relaxation via the sigma38 C-terminal domain.

Escherichia coli responds to stress by a combination of specific and general transcription signalling pathways. The general pathways typically require the master stress regulator sigma38 (rpoS). Here we show that the signalling from multiple stresses that relax DNA is processed by a non-conserved eight-amino-acid tail of the sigma 38 C-terminal domain. By contrast, responses to two stresses that accumulate potassium glutamate do not rely on this short tail, but still require the overall C-terminal domain. In vitro transcription and footprinting studies suggest that multiple stresses can target a poised RNA polymerase and activate it by unwrapping DNA from a nucleosome-like state, allowing the RNA polymerase to escape into productive mode. This transition can be accomplished by either the DNA relaxation or potassium glutamate accumulation that characterizes many stresses.

Regulation of transcription by acetate in Escherichia coli: in vivo and in vitro comparisons.

Acetate, even at neutral pH, induces changes in gene expression that allow Escherichia coli to adapt to the diverse chemical stresses of the gastrointestinal tract. These include differential effects on transcription, including both activation and repression. The in vivo studies presented here show that cyclopropane fatty acid synthase transcription induced by neutral acetate proceeds via both the sigma70 and sigma38 forms of RNA polymerase. Upstream DNA elements are required in both cases, but in vitro studies demonstrate that the binding of regulatory factors is not needed. Sigma70 promoters respond towards acetate with very significant differences in vitro, with effects ranging from inhibition to lack of effect to weak stimulation to strong stimulation. By contrast, the effects of acetate on sigma38 transcription at these same promoters in vitro are generally stimulatory. Thus, acetate directly alters transcription complexes in vitro in ways that lead to significant differential transcription, contributing to the diverse effects that are known to allow adaptation in vivo.

Poising of Escherichia coli RNA polymerase and its release from the sigma 38 C-terminal tail for osmY transcription.

Bacteria must adapt their transcription to overcome the osmotic stress associated with the gastrointestinal tract of their host. This requires the sigma 38 (rpoS) form of RNA polymerase. Here, chromatin immunoprecipitation experiments show that activation is associated with a poise-and-release mechanism in vivo. A C-terminal tail unique among sigma factors is shown to be required for in vivo recruitment of RNA polymerase to the promoter region prior to osmotic shock. C-terminal domain tail-dependent transcription in vivo can be mimicked by using the intracellular signaling molecule potassium glutamate in vitro. Following signaling, the barrier to elongation into the gene body is overcome and RNA polymerase is released to produce osmY mRNA.

Osmolyte-induced transcription: -35 region elements and recognition by sigma38 (rpoS).

In order to meet osmotic challenges in the gastrointestinal tract, enteric bacteria rapidly accumulate salts of glutamate and other weak organic acids. The ensuing transcriptional activation is mediated by unknown elements at sigma38 (rpoS)-dependent promoters. Here we identify DNA elements needed for high levels of transcription in the presence of salt and acetate and show that they are associated with the -35 regions of target promoters. Unrelated -35 region sequences are shown to specify maximal salt-challenged transcription at the otsB promoter and maximal acetate-challenged transcription at the cfa promoter. Mutants in sigma38 are isolated that contribute to bypassing the salt response and most of these cluster in a small segment corresponding to the presumptive -35 DNA recognition determinant of the protein. Overall, the data suggest that an ensemble of -35 region elements exists at sigma38 promoters and these can help mediate responsiveness to physiological challenges through interactions involving region 4 of the sigma38 protein.