Biology subjects

Urem, M.

Publications and source records attributed to Urem, M..

4 recordsLinked to original sources

bioRxiv2025

DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins

Single-stranded DNA-binding proteins (SSBs) play a crucial role in stabilizing and protecting transiently exposed single-stranded DNA (ssDNA), yet the mechanisms governing their displacement by DNA polymerase (DNAp) during replication remain largely unexplored. Using bacteriophage T7 DNAp and its SSB, T7 gp2.5, we investigated the molecular mechanisms and visualized the dynamic process underlying SSB displacement. Our single-molecule force spectroscopy demonstrates that T7 SSB modulates DNA replication in an ssDNA conformation-dependent manner, regulated by tension applied to the DNA template. By integrating dual-color single-molecule imaging, we observe that T7 SSB remains stationary as DNAp approaches, indicating that SSB molecules are sequentially displaced rather than pushed forwards. Molecular dynamics (MD) simulations revealed reduced energy barriers for SSB dissociation in the presence of DNAp. This finding, combined with the detected FRET signals when DNAp approaches an SSB-bound ssDNA region and observations of faster replication rates compared to relative slow intrinsic SSB dissociation, collectively support an active displacement mechanism. Using both ensemble and single-molecule analyses, we demonstrated that SSB saturation of ssDNA is critical for optimal replication efficiency, with each SSB molecule contributing positively to the process. Taken together, the uncovered spatial-temporal coordination between SSB and DNAp is necessary for resolving molecular collisions during DNA replication, and may represent a universal strategy employed by other DNA translocating motors to ensure genomic integrity.

biophysics↗

bioRxiv2024

A new pathway in central metabolism mediates nutrient control of development and antibiotic production by Streptomyces

The amino sugar N-acetylglucosamine (GlcNAc) plays a central role in primary metabolism and is a key signaling molecule for the onset of morphological and chemical differentiation of Streptomyces. The global nutrient-sensory regulator DasR acts as the gatekeeper of development in streptomycetes, and its activity is modulated by aminosugar phosphates. Here, we report the discovery of a novel pathway in aminosugar metabolism that governs GlcNAc sensing. GlcNAc-6P is converted into a toxic metabolite via two new enzyme functions, namely dehydration of N-acetylglucosamine-6-phosphate by NagS to form 6P-Chromogen I, a reaction that has not yet been described in the textbooks, and its subsequent deacetylation by NagA producing a cytotoxic structural analogue of ribose. The latter reveals an unexpected promiscuous activity for GlcNAc-6P deacetylase NagA. The crystal structures of NagS apoenzyme and NagS in complex with its substrate GlcNAc-6P or its inhibitor 6-phosphogluconate were resolved at 2.3 [A], 2.6 [A], and 1.7 [A] resolution, respectively. Detailed in vivo and in vitro studies resolved the key residues of the NagS catalytic site. Thus, we have uncovered a novel pathway in aminosugar metabolism that sheds new light on nutrient-mediated control of development and antibiotic production in Streptomyces.

microbiology↗

bioRxiv2023

Systems-wide analysis of the ROK-family regulatory gene rokL6 and the control of aminosugar toxicity in Streptomyces coelicolor

Streptomycetes are saprophytic bacteria that grow on complex polysaccharides, such as cellulose, starch, chitin and chitosan. For the monomeric building blocks glucose, maltose and N-acetylglucosamine (GlcNAc), the metabolic pathways are well documented, but that of glucosamine (GlcN) is largely unknown. Streptomyces nagB mutants, which lack glucosamine-6-phosphate deaminase activity, fail to grow in the presence of high concentrations of GlcN. Here we report that mutations in the gene for the ROK-family transcriptional regulator RokL6 relieve the toxicity of GlcN in nagB mutants, as a result of elevated expression of the Major Facilitator Superfamily (MFS) exporter SCO1448. Systems- wide analysis using RNA sequencing, ChIP-Seq, EMSAs, 5RACE, bioinformatics and genetics revealed that RokL6 is an autoregulator that represses transcription of sco1448 by binding to overlapping promoters in the rokL6-sco1448 intergenic region. RokL6-independent expression of sco1448 fully relieved toxicity of GlcN to nagB mutants. Taken together, our data show a novel system of RokL6 as a regulator that controls the expression of the MFS transporter SCO1448, which in turn protects cells against GlcN toxicity, most likely by exporting toxic metabolites out of the cell. IMPORTANCECentral metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate (GlcNAc-6P) or glucosamine 6-phosphate (GlcN-6P) is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity". We made use of this concept by selecting for suppressors in genes related to GlcN toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and MFS transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 sheds new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.

microbiology↗

bioRxiv2022

Taxonomic and metabolic diversity of Actinobacteria isolated from faeces of a 28,000-year-old mammoth

Ancient microbial communities of permafrost soils and frozen animal remains represent an archive that has barely been explored. This yet unexplored microbial world is a vast resource that can provide us with new evolutionary insights, metabolic pathways and novel chemistry. Here, we reveal that Actinobacteria isolated from a faecal sample from the intestinal tract of a 28,000-year-old Siberian mammoth are phylogenetically and metabolically distinct from currently known modern siblings. Ancient Micromonospora, Oerskovia, Saccharopolyspora, Sanguibacter and Streptomyces species were successfully revived and their genome sequences resolved. Surprisingly, the genomes of the ancestors show a large phylogenetic distance to strains isolated today and harbour many novel biosynthetic gene clusters that may well represent uncharacterised biosynthetic potential. Metabolic profiles of the strains display production of known molecules like antimycin, conglobatin and macrotetrolides, but the majority of the mass features could not be dereplicated. Our work provides a snapshot into Actinobacteria of the past, yielding unexplored genomic information that is not yet present in current databases.

microbiology↗