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Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans -encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders. An alternative route to CRISPR-induced immunity CRISPR is a microbial RNA-based immune system protecting against viral and plasmid invasions. The CRISPR system is thought to rely on cleavage of a precursor RNA transcript by Cas endonucleases, but not all species with CRISPR-type immunity encode Cas proteins. A new study reveals an alternative pathway for CRISPR activation in the human pathogen Streptococcus pyogenes , in which a trans -encoded small RNA directs processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein. CRISPR is a microbial RNA-based immune system protecting against viral and plasmid invasions. The CRISPR system is thought to rely on cleavage of a precursor RNA transcript by Cas endonucleases, but not all species possessing CRISPR-type immunity encode Cas proteins. This study now describes an alternative pathway in Streptococcus pyogenes that employs trans -encoded small RNA that directs the processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein.

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression 1 , 2 . The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP) 3 – 11 , forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation 12 , 13 and RNA quality control 2 . However, it remains unclear whether transcription–translation coupling—together with its broad functional consequences—is indeed a fundamental characteristic of bacteria other than Escherichia coli . Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis , and that this ‘runaway transcription’ creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP–ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes. In Bacillus subtilis , unlike in Escherichia coli , transcription and translation of genes are not tightly coupled, and pioneering ribosomes lag substantially behind RNA polymerases.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E’s function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide 1 . Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output 2 . However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells 3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5′ ends aligned at transcription start sites and 3′ ends located 200–500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription–translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics. A study reveals that most transcripts in Mycobacterium tuberculosis are incomplete, likely because of the tendency of the transcription machinery in this species to pause on genomic DNA.

Background The transformation of plant photosynthate into soil organic carbon and its recycling to CO 2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon. Results We conducted an experiment in which common wild oats ( Avena fatua) were grown in a 13 CO 2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13 C into their DNA. A 1.45-Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13 C because they preyed upon these bacteria and/or directly from the labeled plant DNA. Isotopic labeling indicates that the population was replicating during the 6-week period of plant growth. Interestingly, the genome is ~ 30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch, and 1,3-β-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate and lactate. The genome also encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to interconvert a variety of isoprenoids, including the plant hormone zeatin. Conclusions Rhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO 2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription. RNA synthesis by bacterial RNA polymerase is interrupted by pauses but their role in RNA synthesis is poorly understood. Here the authors use single-molecule FRET and biochemical analysis to show that pausing regulates branching between the abortive and productive outcomes of initial transcription.

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with fulllength enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specif ic promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N-and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.