Predicting the spread of SARS-CoV-2 variants: An artificial intelligence enabled early detection.

During more than 3 years since its emergence, SARS-CoV-2 has shown great ability to mutate rapidly into diverse variants, some of which turned out to be very infectious and have spread throughout the world causing waves of infections. At this point, many countries have already experienced up to six waves of infections. Extensive academic work has focused on the development of models to predict the pandemic trajectory based on epidemiological data, but none has focused on predicting variant-specific spread. Moreover, important scientific literature analyzes the genetic evolution of SARS-CoV-2 variants and how it might functionally affect their infectivity. However, genetic attributes have not yet been incorporated into existing epidemiological modeling that aims to capture infection trajectory. Thus, this study leverages variant-specific genetic characteristics together with epidemiological information to systematically predict the future spread trajectory of newly detected variants. The study describes the analysis of 9.0 million SARS-CoV-2 genetic sequences in 30 countries and identifies temporal characteristic patterns of SARS-CoV-2 variants that caused significant infection waves. Using this descriptive analysis, a machine-learning-enabled risk assessment model has been developed to predict, as early as 1 week after their first detection, which variants are likely to constitute the new wave of infections in the following 3 months. The model's out-of-sample area under the curve (AUC) is 86.3% for predictions after 1 week and 90.8% for predictions after 2 weeks. The methodology described in this paper could contribute more broadly to the development of improved predictive models for variants of other infectious viruses.

Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

RelA binding of mRNAs modulates translation or sRNA-mRNA basepairing depending on the position of the GGAG site.

Previously, we reported that RelA protein facilitates Hfq-mediated mRNA-sRNA regulation by binding sRNAs carrying a Shine-Dalgarno-like GGAG sequence. In turn, sRNA-Hfq monomers are stabilized, enabling the attachment of more Hfq subunits to form a functional hexamer. Here, using CLIP-seq, we present a global analysis of RelA-bound RNAs showing that RelA interacts with sRNAs as well as with mRNAs carrying a GGAG motif. RelA binding of mRNAs carrying GGAG at position -7 relative to the initiation codon (AUG) inhibits translation by interfering with the binding of 30S ribosomes. The extent of inhibition depends on the distance of GGAG relative to the AUG, as shortening the spacing between GGAG and AUG abrogates RelA-mediated inhibition. Interestingly, RelA binding of target mRNAs carrying GGAG in the coding sequence or close to AUG facilitates target gene regulation by sRNA partners that lack GGAG. However, translation inhibition caused by RelA binding of mRNAs carrying GGAG at position -7 relative to the AUG renders sRNA-mRNA basepairing regulation ineffective. Our study indicates that by binding RNAs carrying GGAG the ribosome-associated RelA protein inhibits translation of specific newly synthesized incoming mRNAs or enables basepairing regulation by their respective sRNA partners, thereby introducing a new regulatory concept for the bacterial response.

RNA binding of Hfq monomers promotes RelA-mediated hexamerization in a limiting Hfq environment.

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.

mRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production.

Cross-Regulation between Bacteria and Phages at a Posttranscriptional Level.

The study of bacteriophages (phages) and prophages has provided key insights into almost every cellular process as well as led to the discovery of unexpected new mechanisms and the development of valuable tools. This is exemplified for RNA-based regulation. For instance, the characterization and exploitation of the antiphage CRISPR (clustered regularly interspaced short palindromic repeat) systems is revolutionizing molecular biology. Phage-encoded proteins such as the RNA-binding MS2 protein, which is broadly used to isolate tagged RNAs, also have been developed as valuable tools. Hfq, the RNA chaperone protein central to the function of many base-pairing small RNAs (sRNAs), was first characterized as a bacterial host factor required for Qß phage replication. The ongoing studies of RNAs are continuing to reveal regulatory connections between infecting phages, prophages, and bacteria and to provide novel insights. There are bacterial and prophage sRNAs that regulate prophage genes, which impact bacterial virulence as well as bacterial cell killing. Conversely, phage- and prophage-encoded sRNAs modulate the expression of bacterial genes modifying metabolism. An interesting subcategory of the prophage-encoded sRNAs are sponge RNAs that inhibit the activities of bacterial-encoded sRNAs. Phages also affect posttranscriptional regulation in bacteria through proteins that inhibit or alter the activities of key bacterial proteins involved in posttranscriptional regulation. However, what is most exciting about phage and prophage research, given the millions of phage-encoded genes that have not yet been characterized, is the vast potential for discovering new RNA regulators and novel mechanisms and for gaining insight into the evolution of regulatory RNAs.

OxyS small RNA induces cell cycle arrest to allow DNA damage repair.

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.

Correction: Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

[This corrects the article DOI: 10.1371/journal.pgen.1005975.].

Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein.

Changes in transcriptional pausing modify the folding dynamics of the pH-responsive RNA element.

Previously, we described a novel pH-responsive RNA element in Escherichia coli that resides in the 5' untranslated region of the alx gene and controls its translation in a pH-dependent manner. Under normal growth conditions, this RNA region forms a translationally inactive structure, but when transcribed under alkaline conditions, it forms an active structure producing the Alx protein. We identified two distinct transcriptional pause sites and proposed that pausing at these sites interfered with the formation of the inactive structure while facilitating folding of the active one. Alkali increases the longevity of pausing at these sites, thereby promoting folding of the translationally active form of alx RNA. We show here that mutations that modify the extent and/or position of pausing, although silent with regard to structure stability per se, greatly influence the dynamics of folding and thereby translation. Our data illustrate the mechanistic design of alx regulation, relying on precise temporal and spatial characteristics. We propose that this unique design provides an opportunity for environmental signals such as pH to introduce structural changes in the RNA and thereby modulate expression.

RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq.

The conserved RNA-binding protein Hfq and its associated small regulatory RNAs (sRNAs) are increasingly recognized as the players of a large network of posttranscriptional control of gene expression in Gram-negative bacteria. The role of Hfq in this network is to facilitate base pairing between sRNAs and their trans-encoded target mRNAs. Although the number of known sRNA-mRNA interactions has grown steadily, cellular factors that influence Hfq, the mediator of these interactions, have remained unknown. We report that RelA, a protein long known as the central regulator of the bacterial-stringent response, acts on Hfq and thereby affects the physiological activity of RyhB sRNA as a regulator of iron homeostasis. RyhB requires RelA in vivo to arrest growth during iron depletion and to down-regulate a subset of its target mRNAs (fdoG, nuoA, and sodA), whereas the sodB and sdhC targets are barely affected by RelA. In vitro studies with recombinant proteins show that RelA enhances multimerization of Hfq monomers and stimulates Hfq binding of RyhB and other sRNAs. Hfq from polysomes extracted from wild-type cells binds RyhB in vitro, whereas Hfq from polysomes of a relA mutant strain shows no binding. We propose that, by increasing the level of the hexameric form of Hfq, RelA enables binding of RNAs whose affinity for Hfq is low. Our results suggest that, under specific conditions and/or environments, Hfq concentrations are limiting for RNA binding, which thereby provides an opportunity for cellular proteins such as RelA to impact sRNA-mediated responses by modulating the activity of Hfq.

A pH-responsive riboregulator.

The locus alx, which encodes a putative transporter, was discovered previously in a screen for genes induced under extreme alkaline conditions. Here we show that the RNA region preceding the alx ORF acts as a pH-responsive element, which, in response to high pH, leads to an increase in alx expression. Under normal growth conditions this RNA region forms a translationally inactive structure, but when exposed to high pH, a translationally active structure is formed to produce Alx. Formation of the active structure occurs while transcription is in progress under alkaline conditions and involves pausing of RNA polymerase at two distinct sites. Alkali increases the longevity of pausing at these sites and thereby interferes with formation of the inactive structure and promotes folding of the active one. The alx locus represents the first example of a pH-responsive riboregulator of gene expression, introducing a novel regulatory mechanism that involves RNA folding dynamics driven by pH.

Stochastic analysis of the SOS response in Escherichia coli.

BACKGROUND: DNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results. METHODOLOGY/PRINCIPAL FINDINGS: Here we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities. CONCLUSION/SIGNIFICANCE: We conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits.

Transient shielding of intimin and the type III secretion system of enterohemorrhagic and enteropathogenic Escherichia coli by a group 4 capsule.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.

Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence.

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed 'genetic islands'. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of approximately 80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the 'genetic islands' of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.

Regulation of gene expression by small non-coding RNAs: a quantitative view.

The importance of post-transcriptional regulation by small non-coding RNAs has recently been recognized in both pro- and eukaryotes. Small RNAs (sRNAs) regulate gene expression post-transcriptionally by base pairing with the mRNA. Here we use dynamical simulations to characterize this regulation mode in comparison to transcriptional regulation mediated by protein-DNA interaction and to post-translational regulation achieved by protein-protein interaction. We show quantitatively that regulation by sRNA is advantageous when fast responses to external signals are needed, consistent with experimental data about its involvement in stress responses. Our analysis indicates that the half-life of the sRNA-mRNA complex and the ratio of their production rates determine the steady-state level of the target protein, suggesting that regulation by sRNA may provide fine-tuning of gene expression. We also describe the network of regulation by sRNA in Escherichia coli, and integrate it with the transcription regulation network, uncovering mixed regulatory circuits, such as mixed feed-forward loops. The integration of sRNAs in feed-forward loops provides tight repression, guaranteed by the combination of transcriptional and post-transcriptional regulations.

Identification of bacterial small non-coding RNAs: experimental approaches.

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed.

Differential regulation of Escherichia coli topoisomerase I by Fis.

We previously reported that the P1 promoter of topA encoding topoisomerase I of Escherichia coli is activated in response to oxidative stress, in a Fis-dependent manner. Here we show that Fis regulation of topA varies with the intracellular concentrations of Fis. Thus, when Fis levels are low, hydrogen peroxide treatment results in topA activation, whereas at high Fis levels hydrogen peroxide treatment renders topA P1 inactive. In vivo DMS footprinting indicates that only at low Fis levels, when exposed to the stress, the region of the topA promoter changes and P1 becomes active. Potassium permanganate experiments indicate that low levels of Fis activate P1 transcription by facilitating the formation of open complexes, while high levels of this protein shut off the promoter. DNase I footprinting show that Fis binds the promoter region of topA at eight sites with different affinities. One low affinity site overlaps the -10, -35 hexamers of RNA polymerase. We propose that in response to oxidative stress, when present at low levels, Fis binds the promoter region of topA at its high affinity sites, thereby facilitating the recruitment of RNA polymerase to P1, while at high levels, Fis occupies the low affinity sites as well, and thus prevents the binding of RNA polymerase. Our results indicate that the oxidative stress response varies in response to changes in growth phase and nutritional environment.

DeoT, a DeoR-type transcriptional regulator of multiple target genes.

In this study, we investigated the genetic organization and function of Escherichia coli yciT, a gene predicted by computational methods to belong to the DeoR-type family of transcriptional regulators. We show that transcription of yciT (here denoted deoT for deoR-Type) initiates from a promoter located upstream of a putative open reading frame (denoted deoL for deoT Leader). We also show that DeoT acts as a global regulator, repressing the expression of a number of genes involved in a variety of metabolic pathways including transport of maltose, fatty acid beta-oxidation and peptide degradation.

Identification of an Escherichia coli operon required for formation of the O-antigen capsule.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.