The bacterial actin homolog MreB is essential for various cellular processes, including cell wall biosynthesis, membrane organization, and cell polarity determination. Given its multifaceted roles, MreB is considered a potential target for antibiotic development. However, the bacterial response to MreB inhibition and the factors contributing to bacterial survival under such conditions are not well understood. In this study, RNA sequencing (RNA-seq) was used to identify genes that are differentially expressed in response to MreB inhibition by the A22 antibiotic or to deletion of the mreBCD operon. We identified 6 upregulated genes and 24 downregulated genes under both conditions. To determine whether the upregulated genes contribute to bacterial survival during MreB inhibition, we performed A22 antibiotic susceptibility assay on mutants deleted for each of the 6 upregulated genes. Our findings reveal that cells lacking DegP, a periplasmic serine protease, are highly suceptible to A22 treatment. Complementation analysis showed that wild-type DegP, but not a protease-defective mutant, mitigated the effects of A22. The morphological defects in DegP-deficient cells caused by A22 were reduced by ectopic expression of related periplasmic proteases, such as DegQ and DegS. Furthermore, elevated temperatures could alleviate the effects of A22 in a DegP-dependent manner. Overall, our study provides a comprehensive analysis of the global transcriptome-wide effects of MreB inhibition, offering new insights into bacterial response to cytoskeleton disruption. The findings highlight the critical role of DegP in bacterial survival during MreB inhibition and suggest potential avenues for developing novel combinatorial antibiotic strategies targeting MreB and DegP. Highlights ### Competing Interest Statement The authors have declared no competing interest. Anusandhan National Research Foundation, CRG/2020/003295 Israel Science Foundation, 1274/19 Wellcome Trust/DBT India Alliance, IA/E/19/1/504958

The crowded intracellular milieu shapes the thermodynamics and kinetics of biochemical reactions. RNA, an abundant and structurally versatile polymer, contributes to this crowding by acting both as a physical agent that restricts diffusion and enhances excluded volume, and as a sequence- and structure-specific scaffold driving multivalent RNA-RNA and RNA-protein interactions. These properties position RNA as a biologically active determinant of intracellular organization, distinct from inert synthetic crowders and purely structural scaffolds. This minireview examines how RNA shapes the formation, composition, and material properties of biomolecular condensates, highlighting the molecular grammar encoded in RNA sequence, length and valency, structure, and chemical modifications. We discuss how concentration-dependent, biphasic effects of RNA on condensate assembly can tip the balance between functional compartmentalization and pathological liquid-to-solid transitions implicated in neurodegenerative disease and cancer. Finally, we outline challenges in defining RNA-specific thresholds and translating structural insights into therapeutic strategies for mitigating aberrant RNA-mediated crowding.

The poles of rod-shaped bacteria emerge as regulatory hubs. We have shown that enzyme I (EI), the major bacterial sugar metabolism regulator, is sequestered when not needed in TmaR phase-separated condensates in Escherichia coli cell poles. Here, we combined genetic and automated microscopy screens to identify residues in EI and TmaR that are important for their interaction and colocalization. Mutating these residues affects EI-TmaR interaction in bacteria and impairs co-phase separation in yeast. The results were used to generate an EI-TmaR interaction model, which agrees with coevolution data and is supported by conservation of the interacting residues and EI-TmaR colocalization in other species. Mutating residues predicted to interact electrostatically further supports our model. The model explains how TmaR controls EI activity and its interaction with the phosphoprotein HPr and, hence, sugar uptake. Our study highlights the importance of sugar metabolism spatial regulation during evolution and presents a way to unravel protein-protein interactions.

Rapid, spatially controlled methods are needed to investigate RNA localization in bacterial cells. APEX2 proximity labeling was shown to be adaptable to rapid RNA labeling in eukaryotic cells and, through the fusion of APEX2 to different proteins targeted to diverse subcellular locations, has been useful to identify RNA localization in these cells. Therefore, we adapted APEX2 proximity labeling of RNA to bacterial cells by generating an APEX2 fusion to the ribonuclease (RNase) E gene, which is necessary and sufficient for bacterial ribonucleoprotein (BR)-body formation. APEX2 fusion is minimally perturbative, and RNA can be rapidly labeled on the sub-minute timescale with alkyne-phenol, outpacing the rapid speed of mRNA decay in bacteria. Alkyne-phenol provides flexibility in the overall application with copper-catalyzed click chemistry for downstream processes, such as fluorescent dye azides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria.

Ribosome profiling has become an essential tool for studying messenger RNA (mRNA) translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency, and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol, we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as those derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.

Bifidobacterium species are well-established members of the human gut microbiome, particularly prominent during infancy, contributing to host health. Within this genus, Bifidobacterium longum ( BL. ) is a widespread species found in both infant and adult guts, known for its complexity and functional diversity among its known subspecies: BL. longum , BL. infantis and BL. suis . Here, using genomic and phylogenetic tools we propose a novel subspecies within the BL. species, Bifidobacterium longum subsp. nexus subspecies novel. We analyzed 435 BL. genomes using a polyphasic taxonomic approach comprising average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), and pangenome analysis. We identified nine BL. strains, isolated from human infants and adults stool samples, as members of a distinct lineage within the BL. species. The type strain, LL6991, was isolated from the stool of a two-week-old Dutch infant in the Lifelines NEXT birth cohort. Phenotypically, BL. nexus exhibits a distinct morphological pattern, predominantly forming rod-shaped cells, often in chains with visible septa, contrasting with the Y-shaped morphology commonly observed for other BL. subspecies. Furthermore, BL. nexus demonstrates unique metabolic capabilities, including efficient utilization of fructose, and starch, carbohydrates not metabolized well by other tested BL. subspecies. This ability may be attributed to specific genes, such as a gene predicted to encode an extracellular amylopullulanase. This characterization expands the known diversity within the BL. species and provides insights into BL. nexus ’s unique adaptations and potential ecological roles within the human gut, especially in infants. Based on the consistent results from genotypic, phylogenetic, and phenotypic analyses, a novel subspecies with the name Bifidobacterium longum subsp. nexus , with type strain LL6991, is proposed. ### Competing Interest Statement T.D.L. is the co-founder and CSO of Microbiotica. Wellcome Trust, 20619, 220540/Z/20/A Netherlands Organization for Scientific Research, VI.C.232.074, NWO Gravitation grant ExposomeNL, 024.004.017 NWO KIC, KICH1.LWV04.21.01 ZonMW ME/CFS, 10091012110015 EU Horizon Europe Program grant INITIALISE, 101094099 EU Horizon Europe Program grant DarkMatter, 101136582

The E. coli transcriptome at the cell’s poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

Liquid-liquid phase separation (LLPS) of proteins was shown in recent years to regulate spatial organization of cell content without the need for membrane encapsulation in eukaryotes and prokaryotes. Yet evidence for the relevance of LLPS for bacterial cell functionality is largely missing. Here we show that the sugar metabolism-regulating clusters, recently shown by us to assemble in the E. coli cell poles by means of the novel protein TmaR, are formed via LLPS. A mutant screen uncovered residues and motifs in TmaR that are important for its condensation. Upon overexpression, TmaR undergoes irreversible liquid-to-solid transition, similar to the transition of disease-causing proteins in human, which impairs bacterial cell morphology and proliferation. Not only does RNA contribute to TmaR phase separation, but by ensuring polar localization and stability of flagella-related transcripts, TmaR enables cell motility and biofilm formation, thus providing a linkage between LLPS and major survival strategies in bacteria.

Hfq, an Sm-like protein and the major RNA chaperone in E. coli, has been shown to distribute non-uniformly along a helical path under normal growth conditions and to relocate to the cell poles under certain stress conditions. We have previously shown that Hfq relocation to the poles is accompanied by polar accumulation of most small RNAs (sRNAs). Here, we show that Hfq undergoes RNA-dependent phase separation to form cytoplasmic or polar condensates of different density under normal and stress conditions, respectively. Purified Hfq forms droplets in the presence of crowding agents or RNA, indicating that its condensation is via heterotypic interactions. Stress-induced relocation of Hfq condensates and sRNAs to the poles depends on the pole-localizer TmaR. Phase separation of Hfq correlates with its ability to perform its posttranscriptional roles as sRNA-stabilizer and sRNA-mRNA matchmaker. Our study offers a spatiotemporal mechanism for sRNA-mediated regulation in response to environmental changes.

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming “RNAP.mRNA.ribosome” complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

The mechanisms controlling membrane recognition by proteins with one hydrophobic stretch at their carboxyl terminus (tail anchor, TA) are poorly defined. The Escherichia coli TAs of ElaB and YqjD, which share sequential and structural similarity with the Saccharomyces cerevisiae TA of Fis1, were shown to localize to mitochondria. We show that YqjD and ElaB are directed by their TAs to bacterial cell poles. Fis1(TA) expressed in E. coli localizes like the endogenous TAs. The yeast and bacterial TAs are inserted in the E. coli inner membrane, and they all show affiliation to phosphatidic acid (PA), found in the membrane of the bacterial cell poles and of the yeast mitochondria. Our results suggest a mechanism for TA membrane recognition conserved from bacteria to mitochondria and raise the possibility that through their interaction with PA, and TAs play a role across prokaryotes and eukaryotes in controlling cell/organelle fate.

EDITORIAL article Front. Microbiol., 29 October 2021Sec. Microbial Physiology and Metabolism https://doi.org/10.3389/fmicb.2021.754109

The omnigenic/polygenic theory, which states that complex traits are not shaped by single/few genes, but by situation-specific large networks, offers an explana-tion for a major enigma in microbiology: deletion of specific small RNAs (sRNAs) playing key roles in various aspects of bacterial physiology, including virulence and antibiotic resistance, results in surprisingly subtle phenotypes. A recent study uncovered polar accumulation of most sRNAs upon osmotic stress, the ma-jority not known to be involved in the applied stress. Here we show that cells deleted for a handful of pole-enriched sRNAs exhibit fitness defect in several stress conditions, as opposed to single, double, or triple sRNA-knockouts, implying that regulation by sRNA relies on sets of genes. Moreover, analysis of RNA-seq data of Escherichia coli and Salmonella typhimurium exposed to antibi-otics and/or infection-relevant conditions reveals the involvement of multiple sRNAs in all cases, in line with the existence of a polygenic plan for sRNA-medi-ated regulation.

Only recently has it been recognized that the transcriptome of bacteria and archaea can be spatiotemporally regulated. All types of prokaryotic transcripts-rRNAs, tRNAs, mRNAs, and regulatory RNAs-may acquire specific localization and these patterns can be temporally regulated. In some cases bacterial RNAs reside in the vicinity of the transcription site, but in many others, transcripts show distinct localizations to the cytoplasm, the inner membrane, or the pole of rod-shaped species. This localization, which often overlaps with that of the encoded proteins, can be achieved either in a translation-dependent or translation-independent fashion. The latter implies that RNAs carry sequence-level features that determine their final localization with the aid of RNA-targeting factors. Localization of transcripts regulates their posttranscriptional fate by affecting their degradation and processing, translation efficiency, sRNA-mediated regulation, and/or propensity to undergo RNA modifications. By facilitating complex assembly and liquid-liquid phase separation, RNA localization is not only a consequence but also a driver of subcellular spatiotemporal complexity. We foresee that in the coming years the study of RNA localization in prokaryotes will produce important novel insights regarding the fundamental understanding of membrane-less subcellular organization and lead to practical outputs with biotechnological and therapeutic implications. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications

RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

Although the list of proteins that localize to the bacterial cell poles is constantly growing, little is known about their temporal behavior. EI, a major protein of the phosphotransferase system (PTS) that regulates sugar uptake and metabolism in bacteria, was shown to form clusters at the Escherichia coli cell poles. We monitored the localization of EI clusters, as well as diffuse molecules, in space and time during the lifetime of E. coli cells. We show that EI distribution and cluster dynamics varies among cells in a population, and that the cluster speed inversely correlates with cluster size. In growing cells, EI is not assembled into clusters in almost 40% of the cells, and the clusters in most remaining cells dynamically relocate within the pole region or between the poles. In non-growing cells, the fraction of cells that contain EI clusters is significantly higher, and dispersal of these clusters is often observed shortly after exiting quiescence. Later, during growth, EI clusters stochastically re-form by assembly of pre-existing dispersed molecules at random time points. Using a fluorescent glucose analog, we found that EI function inversely correlates with clustering and with cluster size. Thus, activity is exerted by dispersed EI molecules, whereas the polar clusters serve as a reservoir of molecules ready to act when needed. Taken together our findings highlight the spatiotemporal distribution of EI as a novel layer of regulation that contributes to the population phenotypic heterogeneity with regard to sugar metabolism, seemingly conferring a survival benefit.

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.