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Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence.
Temperature adaptation of bacterial RNAs is a subject of both fundamental and practical interest because it will allow a better understanding of molecular mechanism of RNA folding with potential industrial application of functional thermophilic or psychrophilic RNAs. Here, we performed a comprehensive study of rRNA, tRNA, and mRNA of more than 200 bacterial species with optimal growth temperatures (OGT) ranging from 4°C to 95°C. We investigated temperature adaptation at primary, secondary and tertiary structure levels. We showed that unlike mRNA, tRNA and rRNA were optimized for their structures at compositional levels with significant tertiary structural features even for their corresponding randomly permutated sequences. tRNA and rRNA are more exposed to solvent but remain structured for hyperthermophiles with nearly OGT-independent fluctuation of solvent accessible surface area within a single RNA chain. mRNA in hyperthermophiles is essentially the same as random sequences without tertiary structures although many mRNA in mesophiles and psychrophiles have well-defined tertiary structures based on their low overall solvent exposure with clear separation of deeply buried from partly exposed bases as in tRNA and rRNA. These results provide new insight into temperature adaptation of different RNAs.
Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being 'capped' at the 5' end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the -1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.
Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.
Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.
Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.
RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.
A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.
CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ∼100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.
Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5' region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.
Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.
Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Repeated extragenic palindromes (REPs) in the enterobacterial genomes are usually composed of individual palindromic units separated by linker sequences. A total of 355 annotated REPs are distributed along the Escherichia coli genome. RNA sequence (RNAseq) analysis showed that almost 80% of the REPs in E. coli are transcribed. The DNA sequence of REP325 showed that it is a cluster of six repeats, each with two palindromic units capable of forming cruciform structures in supercoiled DNA. Here, we report that components of the REP325 element and at least one of its RNA products play a role in bacterial nucleoid DNA condensation. These RNA not only are present in the purified nucleoid but bind to the bacterial nucleoid-associated HU protein as revealed by RNA IP followed by microarray analysis (RIP-Chip) assays. Deletion of REP325 resulted in a dramatic increase of the nucleoid size as observed using transmission electron microscopy (TEM), and expression of one of the REP325 RNAs, nucleoid-associated noncoding RNA 4 (naRNA4), from a plasmid restored the wild-type condensed structure. Independently, chromosome conformation capture (3C) analysis demonstrated physical connections among various REP elements around the chromosome. These connections are dependent in some way upon the presence of HU and the REP325 element; deletion of HU genes and/or the REP325 element removed the connections. Finally, naRNA4 together with HU condensed DNA in vitro by connecting REP325 or other DNA sequences that contain cruciform structures in a pairwise manner as observed by atomic force microscopy (AFM). On the basis of our results, we propose molecular models to explain connections of remote cruciform structures mediated by HU and naRNA4.
The difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome by using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins and exhibit fluorescence increases that enable protein expression to be imaged in living Escherichia coli. These sensors are key components of a generalizable strategy to image protein expression in a single bacterium in real time.
Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
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.
One of the key challenges in computational genomics is annotating coding genes and identification of regulatory RNAs in complete genomes. An attempt is made in this study which uses the regulatory RNA locations and their conserved flanking genes identified within the genomic backbone of template genome to search for similar RNA locations in query genomes. The search is based on recently reported coexistence of small RNAs and their conserved flanking genes in related genomes. Based on our study, 54 additional sRNA locations and functions of 96 uncharacterized genes are predicted in two draft genomes viz., Serratia marcesens Db1 and Yersinia enterocolitica 8081. Although most of the identified additional small RNA regions and their corresponding flanking genes are homologous in nature, the proposed anchoring technique could successfully identify four non-homologous small RNA regions in Y. enterocolitica genome also. The KEGG Orthology (KO) based automated functional predictions confirms the predicted functions of 65 flanking genes having defined KO numbers, out of the total 96 predictions made by this method. This coexistence based method shows more sensitivity than controlled vocabularies in locating orthologous gene pairs even in the absence of defined Orthology numbers. All functional predictions made by this study in Y. enterocolitica 8081 were confirmed by the recently published complete genome sequence and annotations. This study also reports the possible regions of gene rearrangements in these two genomes and further characterization of such RNA regions could shed more light on their possible role in genome evolution.
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