An expanded arsenal of immune systems that protect bacteria from phages.

Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments.

Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution.

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen , analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures.

Global landscape of phenazine biosynthesis and biodegradation reveals species-specific colonization patterns in agricultural soils and crop microbiomes.

Phenazines are natural bacterial antibiotics that can protect crops from disease. However, for most crops it is unknown which producers and specific phenazines are ecologically relevant, and whether phenazine biodegradation can counter their effects. To better understand their ecology, we developed and environmentally-validated a quantitative metagenomic approach to mine for phenazine biosynthesis and biodegradation genes, applying it to >800 soil and plant-associated shotgun-metagenomes.

A 5' UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa.

-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in . There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in , and in doing so we identified AHL-regulated small RNAs (sRNAs).

HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance.

To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, , is a homolog of , which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits.

Bacterial Noncoding RNAs Excised from within Protein-Coding Transcripts.

Prokaryotic genomes encode a plethora of small noncoding RNAs (ncRNAs) that fine-tune the expression of specific genes. The vast majority of known bacterial ncRNAs are encoded from within intergenic regions, where their expression is controlled by promoter and terminator elements, similarly to protein-coding genes. In addition, recent studies have shown that functional ncRNAs can also be derived from gene 3' untranslated regions (3'UTRs) via an alternative biogenesis pathway, in which the ncRNA segment is separated from the mRNA via RNase cleavage.

Successful Brincidofovir Treatment of Metagenomics-detected Adenovirus Infection in a Severely Ill Signal Transducer and Activator of Transcription-1-deficient Patient.

A signal transducer and activator of transcription-1-deficient patient presented with prolonged fever, cachexia, anemia, hypoalbuminemia and finally relapsing debilitating mycobacterial osteomyelitis while receiving a previously effective antimycobacterial treatment. Progression despite rigorous workup and multiple antibiotics prompted shotgun metagenomics revealing adenovirus in liver samples. Brincidofovir led to a complete, sustained clinical recovery, including osteomyelitis, probably attributed to reversal of adenovirus-induced immune dysregulation.

High-resolution RNA 3'-ends mapping of bacterial Rho-dependent transcripts.

Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner.

Extensive reshaping of bacterial operons by programmed mRNA decay.

Bacterial operons synchronize the expression of multiple genes by placing them under the control of a shared promoter. It was previously shown that polycistronic transcripts can undergo differential RNA decay, leaving some genes within the polycistron more stable than others, but the extent of regulation by differential mRNA decay or its evolutionary conservation remains unknown. Here, we find that a substantial fraction of E.

Regulation of antibiotic-resistance by non-coding RNAs in bacteria.

Antibiotic resistance genes are commonly regulated by sophisticated mechanisms that activate gene expression in response to antibiotic exposure. Growing evidence suggest that cis-acting non-coding RNAs play a major role in regulating the expression of many resistance genes, specifically those which counteract the effects of translation-inhibiting antibiotics. These ncRNAs reside in the 5'UTR of the regulated gene, and sense the presence of the antibiotics by recruiting translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA.

Widespread formation of alternative 3' UTR isoforms via transcription termination in archaea.

Transcription termination sets the 3' end boundaries of RNAs and plays key roles in gene regulation. Although termination has been well studied in bacteria, the signals that mediate termination in archaea remain poorly understood. Here, we applied term-seq to comprehensively map RNA 3' termini, with single-base precision, in two phylogenetically distant archaea: Methanosarcina mazei and Sulfolobus acidocaldarius.

Computational prediction of regulatory, premature transcription termination in bacteria.

A common strategy for regulation of gene expression in bacteria is conditional transcription termination. This strategy is frequently employed by 5'UTR cis-acting RNA elements (riboregulators), including riboswitches and attenuators. Such riboregulators can assume two mutually exclusive RNA structures, one of which forms a transcriptional terminator and results in premature termination, and the other forms an antiterminator that allows read-through into the coding sequence to produce a full-length mRNA.

Comparative transcriptomics across the prokaryotic tree of life.

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to 'transcriptional noise'. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life.

Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria.

Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in vivo activity of all such regulators in parallel and enables rapid screening for ribo-regulators that respond to metabolites of choice.

Riboswitches. Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA.

Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor.

Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway.

The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) is the causal agent of speck disease in tomato. Pst pathogenicity depends on a type III secretion system that delivers effector proteins into host cells, where they promote disease by manipulating processes to the advantage of the pathogen.

Expression of Xanthomonas campestris pv. vesicatoria type III effectors in yeast affects cell growth and viability.

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X.