Biology subjects

Meir, M.

Publications and source records attributed to Meir, M..

9 recordsLinked to original sources

bioRxiv2026

Temporal dynamics of RNA phage infection reveal bacterial recovery despite ongoing viral replication

Bacterial defenses against DNA phages are well documented, yet responses to RNA phages remain largely unexplored. Here, we characterize the population dynamics and host responses of Escherichia coli infected by the lytic RNA phage MS2 across multiple timescales, using bulk and single-cell transcriptomics, fluorescence microscopy, whole-genome sequencing, and genetic perturbations. Despite rapid viral takeover and extensive cell death, the bacterial population subsequently rebounds, revealing an unusual state in which host proliferation persists under intense phage pressure. Early in infection, heterogeneous subpopulations are evident, including one with low or undetectable viral transcript levels, low expression of the F-plasmid genes encoding the F-pilus (the MS2 receptor), and a distinct metabolic state. Strikingly, the rebounding population carries MS2 RNA in the great majority of cells, with evidence for active viral replication and continued cell division despite high viral loads. This rebound coincides with strong induction of biofilm-associated genes, including the pga operon, which synthesizes the biofilm-matrix exopolysaccharide PNAG. Deletion of pgaA causes earlier population decline and reduced population survival, indicating that pga-dependent matrix production delays viral lysis. Over longer timescales, phage-exposed populations show heterogeneous F-plasmid prevalence and plasmid-wide expression, consistent with dynamic cycling between plasmid-bearing and plasmid-free states: MS2 selects against F-plasmid-bearing cells, whereas conjugation may restore plasmid carriage. We propose that the biofilm-associated matrix delays viral lysis relative to continued cell division, long enough for rare F-plasmid-free daughter cells to arise and expand. Our results suggest that bacterial survival is determined by a dynamic race between viral lysis and host-cell division.

microbiology↗

bioRxiv2026

Inferring viral proteins that act as public goods during coinfection

Interactions among individuals in structured populations can alter fitness effects of mutations and reshape evolutionary processes. In many systems, including bacteria, yeast, and viruses, such interactions often result in public goods: gene products that are costly to produce yet exploitable by others. During viral coinfection of the same cell, gene products from one genome may complement deleterious mutations in another, allowing defective genomes to persist. Yet it remains difficult to infer which proteins are shareable from population sequencing data, because mutation, selection, drift, and complementation are intertwined. Here, we developed a quantitative framework to infer protein-specific public goods in the RNA bacteriophage MS2, which encodes only four proteins. We analyzed experimental evolution data generated under two multiplicity-of-infection (MOI) regimes: low MOI, where coinfection is rare, and high MOI, where coinfection is common. We first compared empirical mutation patterns between regimes and then applied a Wright-Fisher model combined with simulation-based Bayesian inference using neural posterior estimation. In a two-stage strategy, gene-specific fitness effects were inferred from low-MOI data and subsequently used to estimate protein sharing under high-MOI conditions. Across two statistical inference frameworks, lysis emerged as the strongest public-good candidate, replicase and coat showed an intermediate signal, and maturation showed the weakest evidence for sharing. Together, our results show that viral proteins differ markedly in their propensity to act as public goods. More broadly, they illustrate how coinfection can generate density-dependent selection, a general feature of social evolution that may shape evolutionary dynamics.

evolutionary biology↗

bioRxiv2025

GplR1, an unusual TetR-like transcription factor in Mycobacterium abscessus, controls the production of cell wall glycopeptidolipids, colony morphology, and virulence

Mycobacterium abscessus is a major human pathogen, mostly infecting people with pre-existing lung conditions such as cystic fibrosis. The production of glycopeptidolipids (GPL) is a major determinant of virulence of this bacterium, with clinical isolates that lack GPL generally exhibiting more aggressive clinical behavior. The current paradigm is that GPL production is abolished in vivo via irreversible, spontaneous mutations taking place as part of in-host evolution. Little is known about the mechanisms or extent to which GPL production may be regulated. Here we describe an unusual TetR-like transcription factor of M. abscessus, MAB_1638, that appears to be a strong positive regulator of the entire GPL biosynthesis and export gene cluster through a combination of direct and indirect mechanisms. The inactivation of mab_1638 abolished GPL production and thus led to stable rough colony morphology, as well as increased virulence in infection models, characteristic of rough, non-GPL-producers. Transcriptome analysis found the mab_1638 mutant had 118 differentially expressed genes, including the GPL locus and a second, recently described GPL-like locus that produces a related glycosylated lipopeptide called GP8L. Chromatin Immunoprecipitation and sequencing revealed a consensus inverted-repeat DNA sequence motif characteristic of genes regulated by mab_1638. Together, mab_1638 appears to encode a transcription factor required for production of GPL and therefore having a profound effect on virulence traits. We propose naming this gene GPL regulator 1 (gplR1). This finding raises the important possibility that M. abscessus strains appearing smooth in laboratory growth conditions may nonetheless downregulate GPL-cluster genes in other conditions, including in-patient conditions, and thus acquire the phenotypic characteristics of rough strains. ImportanceMycobacterium abscessus is an important human pathogen, causing disease that is difficult to treat. M. abscessus strains have been observed to have two distinct colony morphologies, smooth and rough, which substantially impact clinical presentation. Rough strains are associated with later-stage, more severe disease and are more virulent in animal models. Smooth morphology is conferred by a molecule called glycopeptidolipid in the outer cell envelope, and rough morphology is known to occur when mutations inactivate genes required for glycopeptidolipid biosynthesis. Little is known about the possibility that glycopeptidolipid production could be regulated. Here we have identified a transcription factor that is required for glycopeptidolipid biosynthesis, indicating that glycopeptidolipid production is indeed a regulated process, and raising the important possibility that strains exhibiting smooth morphology in the lab may down-regulated GPL production in the human host and thereby acquire the virulence properties of rough strains.

microbiology↗

bioRxiv2024

Navigating a fine balance: point-mutant cheater viruses disrupt the viral replication cycle

Cheater viruses, alternatively denoted as defective interfering viruses, cannot replicate on their own yet replicate faster than the wild type (WT) when the two viruses coinfect the same cell. Cheaters must possess dual genetic features: a defect, which leads to their inability to infect cells on their own, and a selective advantage over WT during co-infection. Previously, we have discovered two point-mutant cheaters of the MS2 bacteriophage. Here, we set out to discover the possible repertoire of cheater MS2 viruses by performing experimental evolution at a very high multiplicity of infection (MOI). Our results revealed a third point-mutant cheater that arose in eight biological replicas. Each of the three cheaters disrupts the fine balance necessary for phage replication, in different ways that create a defect + advantage. We found that over time, the point mutant cheaters accumulate additional "helper" mutations, which alter other stages of the viral replication cycle, complementing the disruptions created by the original cheater. Intriguingly, cheater and helper mutations almost always reside in very close proximity on the genome. This region encodes for multiple functions: overlapping reading frames as well as overlapping RNA structures critical for transitioning from one stage to another in the viral replication cycle. This region of overlap explains the dual functions of cheaters, as one mutation can have pleiotropic effects. Overall, these findings underscore how viruses, whose dense genomes often have overlapping functions, can easily evolve point-mutant cheaters, and how cheaters can evolve to alter the intricate balance of the viral replication cycle.

evolutionary biology↗

bioRxiv2023

Mutation rate, selection, and epistasis inferred from RNA virus haplotypes via neural posterior estimation

RNA viruses are particularly notorious for their high levels of genetic diversity, which is generated through the forces of mutation and natural selection. However, disentangling these two forces is a considerable challenge, and this may lead to widely divergent estimates of viral mutation rates, as well as difficulties in inferring fitness effects of mutations. Here, we develop, test, and apply an approach aimed at inferring the mutation rate and key parameters that govern natural selection, from haplotype sequences covering full length genomes of an evolving virus population. Our approach employs neural posterior estimation, a computational technique that applies simulation-based inference with neural networks to jointly infer multiple model parameters. We first tested our approach on synthetic data simulated using different mutation rates and selection parameters while accounting for sequencing errors. Reassuringly, the inferred parameter estimates were accurate and unbiased. We then applied our approach to haplotype sequencing data from a serial-passaging experiment with the MS2 bacteriophage. We estimated that the mutation rate of this phage is around 0.2 mutations per genome per replication cycle (95% highest density interval: 0.051-0.56). We validated this finding with two different approaches based on single-locus models that gave similar estimates but with much broader posterior distributions. Furthermore, we found evidence for reciprocal sign epistasis between four strongly beneficial mutations that all reside in an RNA stem-loop that controls the expression of the viral lysis protein, responsible for lysing host cells and viral egress. We surmise that there is a fine balance between over and under-expression of lysis that leads to this pattern of epistasis. To summarize, we have developed an approach for joint inference of the mutation rate and selection parameters from full haplotype data with sequencing errors, and used it to reveal features governing MS2 evolution.

evolutionary biology↗

bioRxiv2022

The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence

Mycobacterium abscessus causes severe, virtually incurable disease in young patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene expression regulation. Here, we show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough colony morphology, increased pro-inflammatory signaling and virulence in in-vivo infection models, and increased resistance to clinically relevant antibiotics. Examination of clinical isolate cohorts revealed some isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of two mutations found in clinical isolates. Approximate 230 genes were differentially expressed in the B11 deletion mutant. Strains with the clinical B11 mutations showed similar expression trends to the deletion mutant but of a lesser magnitude, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with a model of translational repression via base-pairing of B11 to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences in their RBS, consistent with B11 functioning as a negative regulator through direct binding of target mRNAs. Intriguingly, the genes upregulated in the absence of B11 included components of the ESX-4 secretion system, known to be critical for M. abscessus virulence. One of these genes had a B11-complementary sequence at its RBS, and fusing the UTR of this gene to a reporter was sufficient to make the reporter suppressible by B11. Taken together, our data show that B11 may act as either a negative or positive regulator with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains supports the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. To our knowledge, this is the first report of the role of an sRNA in M. abscessus.

microbiology↗

bioRxiv2021

Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR

Ribonucleic acid (RNA) is a polymer with pivotal functions in many biological processes. RNA structure determination is thus a vital step towards understanding its function. The secondary structure of RNA is stabilized by hydrogen bonds formed between nucleotide base pairs and it defines the positions and shapes of functional stem-loops, internal loops, bulges, and other functional and structural elements. In this work we present a methodology for studying large intact RNA biomolecules using homonuclear 15N solid state nuclear magnetic resonance (NMR) spectroscopy. We show that Proton Driven Spin Diffusion (PDSD) experiments with long mixing times, up to 16s, improved by the incorporation of multiple rotor-synchronous 1H inversion pulses (termed Radiofrequency Dipolar Recoupling, RFDR, pulses), reveal key hydrogen-bond contacts. In the full-length RNA isolated from MS2 phage, we observed strong and dominant contributions of G-C Watson-Crick base pairs, and beyond these common interactions, we observe a significant contribution of the G-U wobble base pairs. Moreover, we can differentiate base-paired and non-base-paired nitrogen atoms. Using the improved technique facilitates characterization of hydrogen-bond types in intact large-scale RNA using solid-state NMR. It can be highly useful to guide secondary structure prediction techniques, and possibly structure determination methods.

biophysics↗

bioRxiv2019

Sequencing Complete Genomes of RNA Viruses With MinION Nanopore: Finding Associations Between Mutations

One of the key challenges in the field of genetics is the inference of haplotypes from next generation sequencing data. The MinION Oxford Nanopore sequencer allows sequencing long reads, with the potential of sequencing complete genes, and even complete genomes of viruses, in individual reads. However, MinION suffers from high error rates, rendering the detection of true variants difficult. Here we propose a new statistical approach named AssociVar, which differentiates between true mutations and sequencing errors from direct RNA/DNA sequencing using MinION. Our strategy relies on the assumption that sequencing errors will be dispersed randomly along sequencing reads, and hence will not be associated with each other, whereas real mutations will display a non-random pattern of association with other mutations. We demonstrate our approach using direct RNA sequencing data from evolved populations of the MS2 bacteriophage, whose small genome makes it ideal for MinION sequencing. AssociVar inferred several mutations in the phage genome, which were corroborated using parallel Illumina sequencing. This allowed us to reconstruct full genome viral haplotypes constituting different strains that were present in the sample. Our approach is applicable to long read sequencing data from any organism for accurate detection of bona fide mutations and inter-strain polymorphisms.

genetics↗

bioRxiv2018

AccuNGS: a sequencing protocol for detection of ultra-rare variants reveals extensive variation in HIV during first days of infection

Mutations fuel evolution and facilitate adaptation to novel environments. However, characterizing the spectrum of mutations in a population is obscured by high error rates of next generation sequencing. Here, we present AccuNGS, a novel in vivo sequencing approach that detects variants as rare as 1:10,000. Applying it to 46 clinical samples taken from early infections of the human-infecting viruses HIV, RSV and CMV, revealed large differences in within-host genetic diversity among virus populations. Haplotype reconstruction revealed that increased diversity was mostly driven by multiple transmitted/founder viruses in HIV and CMV samples. Conversely, we detected an abundance of defective virus genomes (DVGs) in RSV samples, including hyper-edited genomes, nonsense mutations and single point deletions. Higher proportions of DVGs correlated with increased viral loads, suggesting increased cellular co-infection rates, which enable DVG persistence. AccuNGS establishes a general platform that allows detecting DVGs, and in general, rare variants that drive evolution.

bioinformatics↗