RESUMO
Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Animais , Humanos , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Population heterogeneity can promote bacterial fitness in response to unpredictable environmental conditions. A major mechanism of phenotypic variability in the human gut symbiont Bacteroides spp. involves the inversion of promoters that drive the expression of capsular polysaccharides, which determine the architecture of the cell surface. High-throughput single-cell sequencing reveals substantial population heterogeneity generated through combinatorial promoter inversion regulated by a broadly conserved serine recombinase. Exploiting control over population diversification, we show that populations with different initial compositions converge to a similar composition over time. Combining our data with stochastic computational modeling, we demonstrate that the differential rates of promoter inversion are a major mechanism shaping population dynamics. More broadly, our approach could be used to interrogate single-cell combinatorial phase variable states of diverse microbes including bacterial pathogens.
Assuntos
Bactérias , Inversão Cromossômica , Humanos , Regiões Promotoras Genéticas , Bactérias/genética , Polissacarídeos , Análise de Célula ÚnicaRESUMO
Travis et al. (2020) reveal how Francisella tularensis uses stress-induced ppGpp to activate its virulent pathogenesis program by tethering an αCTD-DNA organizer (PigR) to a σ-organizing heterodimer (MglA-SspA), highlighting the remarkable diversity of transcriptional mechanisms in under-studied bacteria.
Assuntos
Francisella tularensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato , Fator sigma/genética , VirulênciaRESUMO
Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.
Assuntos
Transcrição Gênica , Pareamento de Bases/genética , Sequência de Bases , Sequência Consenso/genética , DNA/genética , Cinética , Mutação/genética , Nucleotídeos/metabolismo , RNA/genética , Moldes Genéticos , Elongação da Transcrição GenéticaRESUMO
NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
