Biology subjects

Amster-Choder, O.

Publications and source records attributed to Amster-Choder, O..

5 recordsLinked to original sources

bioRxiv2026

Periplasmic proteostasis enables bacterial survival during MreB cytoskeletal disruption

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. HighlightsO_LIMreB disruption by A22 or mutation causes major transcriptome alterations C_LIO_LISix genes are consistently upregulated under both MreB disruption conditions C_LIO_LIDegP, a periplasmic protease, is crucial for bacterial tolerance to A22 C_LIO_LIElevated temperatures alleviate A22 toxicity in a DegP-dependent manner C_LI

microbiology↗

bioRxiv2025

Bifidobacterium longum subsp. nexus nov., a novel subspecies isolated from infant stool

Bifidobacterium longum is a foundational, genetically diverse member of the infant gut microbiome, yet resolving its subspecies in metagenomic data remains challenging. Here, we performed a deep phylogenetic characterization of the B. longum species utilizing 4,577 fecal metagenomes from the LLNEXT mother-infant cohort alongside a global dataset of 1,220 genomes and metagenome-assembled genomes (MAGs). We identified a novel, geographically widespread lineage: Bifidobacterium longum subsp. nexti. Using a polyphasic taxonomic analysis, including average nucleotide identity and digital DNA-DNA hybridization, confirms subspecies-level divergence. Distinct from other members of the B. longum species, BL. nexti harbors -amylopullulanase and grows on soluble starch and often pullulan, consistent with -glucan utilization. Within the LLNEXT cohort, BL. nexti is enriched in formula-fed infants and shows evidence consistent with vertical transmission. Globally, while BL. nexti is geographically widespread, it usually persists at low relative abundance across diverse populations. By resolving a major fraction of previously unclassified B. longum diversity, this study provides new insights into the specialized lineages and ecological dynamics driving the assembly of the developing infant microbiome.

microbiology↗

bioRxiv2025

A simple, fast and cost-efficient protocol for ultra-sensitive ribosome profiling

Ribosome profiling has become an essential tool for studying 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 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.

systems biology↗

bioRxiv2022

Regulation of major bacterial survival strategies by transcript sequestration in a membraneless organelle

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.

microbiology↗

bioRxiv2020

Tyrosine phosphorylation-dependent localization of TmaR, a novel E. coli polar protein that controls activity of the major sugar regulator by polar sequestration

The poles of E. 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 novel 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 of the general PTS protein Enzyme I (EI) by preventing it from exerting its activity by binding and polar sequestration, 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 argue 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 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. SIGNIFICANCEIn recent years, we have learnt that bacterial cells have intricate spatial organization despite the lack of membrane-bounded organelles. The endcaps of rod-shaped bacteria, termed poles, are emerging as hubs for sensing and responding, but the underlying mechanisms for positioning macromolecules there are largely unknown. We discovered a novel protein, TmaR, whose polar localization depends on a phospho-tyrosine modification. We show that TmaR controls the activity of EI, the major regulator of sugar metabolism in most bacteria, by polar sequestration and release. Notably, TmaR is essential for survival in conditions that E. coli often encounters in nature. Hence, TmaR is a key regulator that connects tyrosine phosphorylation, spatial regulation, sugar metabolism and survival in bacteria and the first protein reported to recruit proteins to the E. coli cell poles.

microbiology↗