Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also found that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also found that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Tuberculosis treatment failure associated with evolution of antibiotic resilience.

The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis (Mtb)clinical isolates has identified an essential transcriptional regulator, Rv1830, herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

Escherichia coli SymE is a DNA-binding protein that can condense the nucleoid.

Type I toxin-antitoxin (TA) systems typically consist of a protein toxin that imbeds in the inner membrane where it can oligomerize and form pores that change membrane permeability, and an RNA antitoxin that interacts directly with toxin mRNA to inhibit its translation. In Escherichia coli, symE/symR is annotated as a type I TA system with a non-canonical toxin. SymE was initially suggested to be an endoribonuclease, but has predicted structural similarity to DNA binding proteins. To better understand SymE function, we used RNA-seq to examine cells ectopically producing it. Although SymE drives major changes in gene expression, we do not find strong evidence of endoribonucleolytic activity. Instead, our biochemical and cell biological studies indicate that SymE binds DNA. We demonstrate that the toxicity of symE overexpression likely stems from its ability to drive severe nucleoid condensation, which disrupts DNA and RNA synthesis and leads to DNA damage, similar to the effects of overproducing the nucleoid-associated protein H-NS. Collectively, our results suggest that SymE represents a new class of nucleoid-associated proteins that is widely distributed in bacteria.

Global Analysis of the Specificities and Targets of Endoribonucleases from Escherichia coli Toxin-Antitoxin Systems.

Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the targets of most of these toxins remain poorly characterized. Using a new RNA sequencing (RNA-seq) pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterized the targets and specificities of 9 endoribonuclease toxins from E. coli. We found that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli, but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis. IMPORTANCE Toxin-antitoxin (TA) systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacterized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets. Using a new and broadly applicable RNA-seq pipeline, we carried out a global analysis of 9 endoribonuclease toxins from E. coli. We found that each uses a relatively low-information cleavage motif to cut a large proportion of mRNAs in E. coli, but not tRNAs or mature rRNAs. Notably, although the precise set of targets varies, each toxin efficiently disrupts ribosome biogenesis, primarily by cleaving the mRNAs of ribosomal proteins. In sum, the analyses presented provide new, comprehensive insights into the cleavage specificities and targets of almost all endoribonuclease toxins in E. coli. Despite different specificities, our work reveals a striking commonality in function, as each toxin disrupts ribosome biogenesis and translation.

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics-even below this breakpoint-is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.

Stress Can Induce Transcription of Toxin-Antitoxin Systems without Activating Toxin.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.

A Simple, Cost-Effective, and Robust Method for rRNA Depletion in RNA-Sequencing Studies.

The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

Global Analysis of the E. coli Toxin MazF Reveals Widespread Cleavage of mRNA and the Inhibition of rRNA Maturation and Ribosome Biogenesis.

Toxin-antitoxin systems are widely distributed genetic modules that regulate growth and persistence in bacteria. Many systems, including E. coli MazEF, include toxins that are endoribonucleases, but the full set of targets for these toxins remains poorly defined. Previous studies on a limited set of transcripts suggested that MazF creates a pool of leaderless mRNAs that are preferentially translated by specialized ribosomes created through MazF cleavage of mature 16S rRNA. Here, using paired-end RNA sequencing (RNA-seq) and ribosome profiling, we provide a comprehensive, global analysis of MazF cleavage specificity and its targets. We find that MazF cleaves most transcripts at multiple sites within their coding regions, with very few full-length, leaderless mRNAs created. Additionally, our results demonstrate that MazF does not create a large pool of specialized ribosomes but instead rapidly disrupts ribosome biogenesis by targeting both ribosomal protein transcripts and rRNA precursors, helping to inhibit cell growth.

Electrostatic effect of the ribosomal surface on nascent polypeptide dynamics.

The crucial molecular events accompanying protein folding in the cell are still largely unexplored. As nascent polypeptides emerge from the ribosomal exit tunnel, they come in close proximity with the highly negatively charged ribosomal surface. How is the nascent polypeptide influenced by the ribosomal surface? We address this question via the intrinsically disordered protein PIR and a number of its variably charged mutants. Two different populations are identified: one is highly spatially biased, and the other is highly dynamic. The more negatively charged nascent polypeptides emerging from the ribosome are richer in the extremely dynamic population. Hence, nascent proteins with a net negative charge are less likely to interact with the ribosome. Surprisingly, the amplitude of the local motions of the highly dynamic population is much wider than that of disordered polypeptides under physiological conditions, implying that proximity to the ribosomal surface enhances the molecular flexibility of a subpopulation of the nascent protein, much like a denaturing agent would. This effect could be important for a proper structural channeling of the nascent protein and the prevention of cotranslational kinetic trapping. Interestingly, a significant population of the highly spatially biased nascent chain, probably interacting extensively with the ribosome, is present even for very negatively charged nascent proteins. This "sticking" effect likely serves to protect nascent proteins (e.g., from cotranslational aggregation). In all, our results highlight the influence of the ribosome in nascent protein dynamics and show that the ribosome's function in protein biogenesis extends well beyond catalysis of peptide bond formation.

Production of ribosome-released nascent proteins with optimal physical properties.

The growing interest in protein folding under physiologically relevant conditions has prompted investigations requiring direct comparisons between ribosome-bound and ribosome-released nascent proteins. Such studies, involving the ad hoc release of newly synthesized proteins from stalled ribosomes, demand a release agent able to produce nonaggregated native proteins and preserve the overall nature of the medium. Here, we explore hydroxylamine, a reactant rarely used to release nascent chains, and compare it to other ribosome-release agents: puromycin, RNase A/EDTA, and sodium hydroxide. Ribosome-bound nascent chains corresponding to the sequence of apoHmpH, the Escherichia coli N-terminal domain of Hmp, were used as a model system. Fluorescence anisotropy decays were employed to probe the self-association and overall physical properties of nascent proteins. Gel electrophoresis and RNA chip microfluidic capillary electrophoresis yielded information on the integrity of nascent peptidyl-tRNAs and ribosomes, respectively. Of the four reagents examined, only hydroxylamine releases nascent apoHmpH without causing extensive aggregation or degradation of the ribosome. Hydroxylamine does not introduce large hydrophobic C-terminal modifications and functions at nearly physiological pH. It is therefore a suitable reagent for the ad hoc release of nascent proteins from the ribosome.

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions.

We still know very little about how proteins achieve their native three-dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton-sized ribosome pose special challenges due to the large size and complicated nature of the ribosome-nascent chain complex. This work introduces a combination of three-component analysis of fluorescence depolarization decays (including the presence of two local motions) and in-cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all-alpha-helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome-nascent chain complexes. We find that the sub-ns motions of the N-terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high-order parameters (>0.85) and small cone semiangles (<30 degrees ). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.