High-throughput transcriptomics of 409 bacteria-drug pairs reveals drivers of gut microbiota perturbation.

Many drugs can perturb the gut microbiome, potentially leading to negative health consequences. However, mechanisms of most microorganism-drug responses have not been elucidated at the genetic level. Using high-throughput bacterial transcriptomics, we systematically characterized the gene expression profiles of prevalent human gut bacteria exposed to the most frequently prescribed orally administered pharmaceuticals. Across >400 drug-microorganism pairs, significant and reproducible transcriptional responses were observed, including pathways involved in multidrug resistance, metabolite transport, tartrate metabolism and riboflavin biosynthesis. Importantly, we discovered that statin-mediated upregulation of the AcrAB-TolC efflux pump in Bacteroidales species enhances microbial sensitivity to vitamin A and secondary bile acids. Moreover, gut bacteria carrying acrAB-tolC genes are depleted in patients taking simvastatin, suggesting that drug-efflux interactions generate collateral toxicity that depletes pump-containing microorganisms from patient microbiomes. This study provides a resource to further understand the drivers of drug-mediated microbiota shifts for better informed clinical interventions.

Bacterial genome engineering using CRISPR-associated transposases.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases 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 Escherichia coli at efficiencies approaching ~100%. Moreover, they 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 CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1-2 weeks using available plasmid constructs and standard molecular biology techniques.

Conversion of Ribosome-Protected mRNA to DNA and Deep Sequencing for Ribosome Profiling in Haloferax volcanii.

The translation of messenger RNA (mRNA) into protein is an essential process for all forms of life. The ability to monitor this process in a quantitative way by ribosome profiling-based approaches has revolutionized our ability to monitor protein synthesis in vivo and to explore and model complex cellular processes. Ribosome profiling is a high-throughput technique that globally analyzes the full set of ribosomes engaged in translation, providing insights into important aspects of the mechanism of protein synthesis and its regulation. This protocol covers the construction of a ribosome profiling library from culture harvesting, footprint isolation via ultracentrifugation, gel-based size fractionation, and footprint sequencing for a model halophilic archaeon, Haloferax volcanii. This approach has revealed the first global view of translation in the archaea.

Small RNA-Sequencing Library Preparation for the Halophilic Archaeon Haloferax volcanii.

Posttranscriptional regulation actuated by small RNAs (sRNAs) plays essential roles in a wide variety of cellular processes, especially in stress responses and environmental signaling. Hundreds of sRNAs have recently been discovered in archaea using genome-wide approaches but the molecular mechanisms of only a few have been characterized experimentally. Here, we describe how to build sRNA sequencing libraries using size-selected total RNA in the model archaeon, Haloferax volcanii , to provide a tool to further characterize sRNAs in archaea.

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea.

While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volc anii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative 'seed' region within the predicted stem loop of SHOxi. Malic enzyme catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homoeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution.

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

The Non-Coding Regulatory RNA Revolution in Archaea.

Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Recently, high throughput studies using RNA-sequencing revealed that sRNAs are broadly expressed in the Archaea, comprising thousands of transcripts within the transcriptome during non-challenged and stressed conditions. Antisense sRNAs, which overlap a portion of a gene on the opposite strand (cis-acting), are the most abundantly expressed non-coding RNAs and they can be classified based on their binding patterns to mRNAs (3' untranslated region (UTR), 5' UTR, CDS-binding). These antisense sRNAs target many genes and pathways, suggesting extensive roles in gene regulation. Intergenic sRNAs are less abundantly expressed and their targets are difficult to find because of a lack of complete overlap between sRNAs and target mRNAs (trans-acting). While many sRNAs have been validated experimentally, a regulatory role has only been reported for very few of them. Further work is needed to elucidate sRNA-RNA binding mechanisms, the molecular determinants of sRNA-mediated regulation, whether protein components are involved and how sRNAs integrate with complex regulatory networks.

Transcriptional Landscape and Regulatory Roles of Small Noncoding RNAs in the Oxidative Stress Response of the Haloarchaeon Haloferax volcanii.

Haloarchaea in their natural environment are exposed to hypersalinity, intense solar radiation, and desiccation, all of which generate high levels of oxidative stress. Previous work has shown that haloarchaea are an order of magnitude more resistant to oxidative stress than most mesophilic organisms. Despite this resistance, the pathways haloarchaea use to respond to oxidative stress damage are similar to those of nonresistant organisms, suggesting that regulatory processes might be key to their robustness. Recently, small regulatory noncoding RNAs (sRNAs) were discovered in Archaea under a variety of environmental conditions. We report here the transcriptional landscape and functional roles of sRNAs in the regulation of the oxidative stress response of the model haloarchaeon Haloferax volcanii Thousands of sRNAs, both intergenic and antisense, were discovered using strand-specific sRNA sequencing (sRNA-seq), comprising 25 to 30% of the total transcriptome under no-challenge and oxidative stress conditions, respectively. We identified hundreds of differentially expressed sRNAs in response to hydrogen peroxide-induced oxidative stress in H. volcanii The targets of a group of antisense sRNAs decreased in expression when these sRNAs were upregulated, suggesting that sRNAs are potentially playing a negative regulatory role on mRNA targets at the transcript level. Target enrichment of these antisense sRNAs included mRNAs involved in transposon mobility, chemotaxis signaling, peptidase activity, and transcription factors.IMPORTANCE While a substantial body of experimental work has been done to uncover the functions of small regulatory noncoding RNAs (sRNAs) in gene regulation in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in the haloarchaeon Haloferax volcanii Our work demonstrates that common principles for the response to a major cellular stress exist across the 3 domains of life while uncovering pathways that might be specific to the Archaea This work also underscores the relevance of sRNAs in adaptation to extreme environmental conditions.