Molecular model of a bacterial flagellar motor reveals a "parts-list" of protein adaptations to increase torque.

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures . Here we describe a minicell system that enables cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque motor including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque.

Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs).

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen .

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen .

Revealing the small proteome of by combining ribosome profiling and small-protein optimized mass spectrometry.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins.

Unraveling the small proteome of the plant symbiont by ribosome profiling and proteogenomics.

The soil-dwelling plant symbiont is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology.

RNA recording in single bacterial cells using reprogrammed tracrRNAs.

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.

Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.

CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear.

RiboReport - benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria.

Small proteins encoded by short open reading frames (ORFs) with 50 codons or fewer are emerging as an important class of cellular macromolecules in diverse organisms. However, they often evade detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation in genomic regions previously thought to be non-coding, driving the development of ORF detection tools using Ribo-seq data.

RNase III-mediated processing of a -acting bacterial sRNA and its -encoded antagonist.

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes.

Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria.

Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution.

Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq.

CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome.

The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR-Cas immune systems.

CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A).

Identifying Small Open Reading Frames in Prokaryotes with Ribosome Profiling.

Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes.

Small RNA mediated gradual control of lipopolysaccharide biosynthesis affects antibiotic resistance in Helicobacter pylori.

The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach.

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from , we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9.

Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies.

Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE-respiring representatives of the genus, uncovered the genetic inactivation of a two-component system (TCS) in the OHR gene region of the natural mutants.

HRIBO: high-throughput analysis of bacterial ribosome profiling data.

Motivation: Ribosome profiling (Ribo-seq) is a powerful approach based on deep sequencing of cDNA libraries generated from ribosome-protected RNA fragments to explore the translatome of a cell, and is especially useful for the detection of small proteins (50-100 amino acids) that are recalcitrant to many standard biochemical and in silico approaches. While pipelines are available to analyze Ribo-seq data, none are designed explicitly for the automatic processing and analysis of data from bacteria, nor are they focused on the discovery of unannotated open reading frames (ORFs).

Results: We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data.

A global data-driven census of small proteins and their potential functions in bacterial virulence.

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than . We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Typhimurium.

A Repeat-Associated Small RNA Controls the Major Virulence Factors of Helicobacter pylori.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter.

Differences in the Transcriptomic Response of and to Heat Stress.

spp. are one of the most important food-borne pathogens, which are quite susceptible to environmental or technological stressors compared to other zoonotic bacteria. This might be due to the lack of many stress response mechanisms described in other bacteria.

A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host.

Structural insights into the AapA1 toxin of Helicobacter pylori.

Background: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear.

Methods: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization.

A non-coding RNA from the intercellular adhesion (ica) locus of Staphylococcus epidermidis controls polysaccharide intercellular adhesion (PIA)-mediated biofilm formation.

Polysaccharide intercellular adhesin (PIA)-associated biofilm formation is mediated by the intercellular adhesin (ica) locus and represents a major pathomechanism of Staphylococcus epidermidis. Here, we report on a novel long non-coding (nc)RNA, named IcaZ, which is approximately 400 nucleotides in size. icaZ is located downstream of the ica repressor gene icaR and partially overlaps with the icaR 3' UTR.

The small non-coding RNA RsaE influences extracellular matrix composition in Staphylococcus epidermidis biofilm communities.

RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S.