Bacterial RNA degradosomes are multienzyme molecular machines that act as hubs for posttranscriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation.In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries and their similarities with the eukaryotic mRNP granules.

Toxin−antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin−antitoxin system (AapA1−IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori. We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5′-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.

Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity. IMPORTANCE Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.

Motivation With the steadily increasing abundance of omics data produced all over the world under vastly different experimental conditions residing in public databases, a crucial step in many data-driven bioinformatics applications is that of data integration. The challenge of batch-effect removal for entire databases lies in the large number of batches and biological variation which can result in design matrix singularity. This problem can currently not be solved satisfactorily by any common batch-correction algorithm. Results We present reComBat, a regularized version of the empirical Bayes method to overcome this limitation and benchmark it against popular approaches for the harmonization of public gene expression data (both microarray and bulkRNAsq) of the human opportunistic pathogen Pseudomonas aeruginosa. Batch-effects are successfully mitigated while biologically meaningful gene expression variation is retained. reComBat fills the gap in batch-correction approaches applicable to large-scale, public omics databases and opens up new avenues for data-driven analysis of complex biological processes beyond the scope of a single study. Availability The code is available at https://github.com/BorgwardtLab/reComBat, all data and evaluation code can be found at https://github.com/BorgwardtLab/batchCorrectionPublicData

Importance 46Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of 47 half of the human population worldwide. Infection by H. pylori can lead to the 48 development of gastric pathologies such as ulcers and adenocarcinoma, that causes 49 up to 800.000 deaths in the world each year. Persistent colonization by H. pylori relies 50 on regulation of the expression of adaptation-related genes. One major level of such 51 control is post-transcriptional regulation that, in H. pylori, largely relies on a multi-52 protein molecular machine, an RNA-degradosome, that we previously discovered. In 53 this study, we established that the two protein partners of this machine are associated 54 to the membrane of H. pylori. Using cutting-edge microscopy, we showed that these 55 complexes assemble into hubs whose formation is regulated by free RNA and scaled 56 with bacterial size and growth phase. Cellular compartmentalization of molecular 57 machines into hubs emerges as an important regulatory level in the organelle-less 58 bacteria. 59 61Post-transcriptional regulation is one of the most important levels of control of gene 62 expression in every kingdom of life. Ribonucleases (RNases) are key enzymes in post-63 transcriptional regulation, involved in RNA maturation and degradation. RNases often 64 act in multi-protein complexes that are designated exosomes in Eukarya and Archaea 65 and RNA degradosomes in bacteria and chloroplasts [for a review, see (1)]. RNA-66 degradosomes were established in several bacterial species and are defined by two 67 core components, an RNase and an RNA helicase (2). These RNA helicases belong 68 to the DEAD-box family and act by unwinding RNA, thereby allowing access of the 69 ribonucleases to some of their target sites on RNAs. RNA degradosomes are 70 widespread and vary in composition, although only few have been described in detail 71(1). Most RNA degradosomes reported so far are assembled on the essential 72 endoribonuclease RNase E, like in Escherichia coli, Caulobacter crescentus or 73 Mycobacterium tuberculosis (3-5). In the E. coli degradosome, RNase E serves as a 74 scaffold for the binding of the DEAD-box RNA helicase RhlB, the metabolic enzyme 75 enolase and the 3'-5' exoribonuclease PNPase (2, 6). Nevertheless, our recent 76 analysis on a representative set of 1,535 bacterial genomes revealed that RNase E is 77 absent from about half of the bacterial species (1). Most of the remaining bacteria 78 (47%), that lack RNase E, have either RNase Y or RNase J enzymes or both. RNase 79 J and RNase Y, first identified in Bacillus subtilis, both display endoribonucleolytic 80 activities but RNase J acts in addition as a 5'-3' exoribonuclease (7, 8). Although being 81 unrelated proteins, these enzymes constitute functional homologues of RNase E. In 82 the Gram-positive bacteria Staphylococcus aureus and B. subtilis, RNA 83 degradosomes comprising RNase Y and RNase J have been proposed, but to date 84 20). Interestingly, similarly to the situation in H. pylori, the E. ...

Helicobacter pylori is a Gram-negative bacterial pathogen that colonizes the stomach of about half of the human population worldwide. Infection by H. pylori is generally acquired during childhood and this bacterium rapidly establishes a persistent colonization. H. pylori causes chronic gastritis that, in some cases, progresses into peptic ulcer disease or adenocarcinoma that is responsible for about 800,000 deaths in the world every year. H. pylori has evolved efficient adaptive strategies to colonize the stomach, a particularly hostile acidic environment. Few transcriptional regulators are encoded by the small H. pylori genome and post-transcriptional regulation has been proposed as a major level of control of gene expression in this pathogen. The transcriptome and transcription start sites (TSSs) of H. pylori strain 26695 have been defined at the genome level. This revealed the existence of a total of 1,907 TSSs among which more than 900 TSSs for non-coding RNAs (ncRNAs) including 60 validated small RNAs (sRNAs) and abundant anti-sense RNAs, few of which have been experimentally validated. An RNA degradosome was shown to play a central role in the control of mRNA and antisense RNA decay in H. pylori. Riboregulation, genetic regulation by RNA, has also been revealed and depends both on antisense RNAs and small RNAs. Known examples will be presented in this review. Antisense RNA regulation was reported for some virulence factors and for several type I toxin antitoxin systems, one of which controls the morphological transition of H. pylori spiral shape to round coccoids. Interestingly, the few documented cases of small RNA-based regulation suggest that their mechanisms do not follow the same rules that were well established in the model organism Escherichia coli. First, the genome of H. pylori encodes none of the two well-described RNA chaperones, Hfq and ProQ that are important for riboregulation in several organisms. Second, some of the reported small RNAs target, through “rheostat”-like mechanisms, repeat-rich stretches in the 5′-untranslated region of genes encoding important virulence factors. In conclusion, there are still many unanswered questions about the extent and underlying mechanisms of riboregulation in H. pylori but recent publications highlighted original mechanisms making this important pathogen an interesting study model.

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

25 26 suggesting coccoid viability. Using single-cell live microscopy, we observed that shape 42 conversion is associated with cell division interference. Oxidative stress represses antitoxin 43 promoter activity and enhances processing of its transcript leading to an imbalanced ratio in 44 favor of AapA1 toxin expression. 45Our data are in favor of viable coccoids with characteristics of dormant bacteria that might 46 be important in H. pylori infections refractory to treatment. 47 Significance Statement 49Helicobacter pylori, a gastric pathogen responsible for 800,000 deaths in the world every 50year, is encountered, both in vitro and in patients, as spiral-shaped bacteria and as round cells 51 named coccoids. We discovered that the toxin from a chromosomal type I toxin-antitoxin 52 system is targeting H. pylori membrane and acting as an effector of the morphological 53 conversion of H. pylori to coccoids. We showed that these round cells maintain their 54 membrane integrity and metabolism, strongly suggesting that they are viable dormant bacteria. 55Oxidative stress was identified as a signal inducing toxin expression. Our findings reveal new 56 insights into a form of dormancy of this bacterium that might be associated with H. pylori 57 infections refractory to treatment. 58 59 60 (10) and it was more generally found that decreased intracellular ATP concentration is a 79 landmark of persister formation (11). 80In the present work, we explored the role of TA systems in Helicobacter pylori, a 81 bacterium that colonizes the stomach of half of the human population worldwide and causes 82 the development of gastritis. In some cases, gastritis evolves into peptic ulcer disease or 83 gastric carcinoma that causes about 800,000 deaths in the world every year (12, 13). This 84 microaerophilic bacterium is unique in its capacity to persistently colonize the stomach 85 despite its extreme acidity and intense immune response (13). The molecular mechanisms at 86 both translation inhibition of the AapA1 active message and leading to rapid degradation of 104 the duplex by RNase III has been recently published (20). The H. pylori type I toxins are 105 typically small hydrophobic peptides of 30-40 amino acids predicted to form alpha-helices. 106No clues on the mode of action or the physiological role of these systems have been reported. 107Here we show that the AapA1 toxin induces a rapid and massive morphological 108 transformation of H. pylori from spirals to coccoids by targeting the inner membrane and 109 interfering with cell division, and that oxidative stress triggers imbalanced expression of the 110 TA components in favor of toxin production. 111 Results 112IsoA1 or pA1*. In contrast, addition of the inducer causes a rapid and immediate growth 129 arrest of H. pylori with pA1 indicating a toxic effect of AapA1 expression (Fig. 1B). The 130 growth arrest was accompanied by loss of culturability of more than 10 4 -fold 8h after 131 induction. 132In parallel to growth, we investigated the consequences of AapA1 expressio...