Biology subjects

Dame, R. T.

Publications and source records attributed to Dame, R. T..

13 recordsLinked to original sources

bioRxiv2026

Context-dependent regulation of RpoS-dependent and RpoS-independent gene transcription by Crl during exponential growth in Escherichia coli

RpoS, an alternative sigma factor, directly regulates approximately 10% of gene transcription in E. coli. The small protein Crl functions as a partner that assists RpoS in exerting its regulatory role, particularly during the stationary phase. However, the coordinating role of Crl during the exponential phase remains unclear. In addition, whether Crl can regulate gene transcription independently of RpoS is still unknown. Therefore, in this study, we used the RpoS-dependent operon hdeAB and the RpoS-independent operon proVWX as model systems to investigate the transcriptional regulatory function of Crl using RT-qPCR and 3C-qPCR. Since the expression of both Crl and RpoS is modulated by environmental signals, we further examined the transcription of these operons under different pH and osmotic conditions. The results showed that Crl can regulate transcription independently of RpoS and exhibits environment-specific bidirectional regulation. Overall, this study improves our understanding of the regulatory mechanisms of Crl and highlights the complexity of transcriptional regulation.

microbiology↗

bioRxiv2026

Adaptive gene transcription in Escherichia coli under environmental stress

Escherichia coli is highly sensitive to acid and osmotic stress but adapts by modulating the expression of stress responsive genes. Nucleoid-associated proteins (NAPs) play key roles in DNA organization and sensing environmental changes. The histone-like nucleoid structuring protein H-NS is an NAP acting as a global regulator of stress genes. H-NS may alter local chromatin structure to modulate the expression of such genes in response to environmental stress. The H-NS homolog StpA co-regulates several target genes, but its precise role is poorly defined. To investigate the regulatory interplay between these two proteins, we examined transcription, DNA binding and chromatin structure at two regulated operons, hdeAB and proVWX, in E. coli following exposure to acid and salt shock. Our results show that H-NS senses pH and osmotic cues to remodel chromatin and relieve repression, while StpA compensates for H-NS loss, particularly at proVWX, highlighting a coordinated regulatory network.

microbiology↗

bioRxiv2025

Archaeal histone HTkC hypercompacts DNA

Histones are important organizers of chromatin in eukaryotes and archaea. In eukaryotes, the core histones assemble with DNA to form the octameric nucleosome. In archaea, histones form hypernucleosomes that are not restricted to an octameric histone core but can extend to variable lengths. We previously identified face-to-face (FtF) histones as a widely distributed group of archaeal histones that assemble into toroidal tetramer structures, distinct from nucleosomal histones. Here, we characterize the FtF histone HTkC from Thermococcus kodakarensis, which also encodes the canonical histones HTkA and HTkB. We show that HTkC wraps DNA around its toroidal tetramer and forms highly compact nucleoprotein complexes, achieving a level of compaction approximately twice that of hypernucleosomes. Consistent with a major chromatin-organizing role, htkC is among the most highly expressed genes in T. kodakarensis and its deletion leads to impaired growth. Together, these findings establish FtF histones as important organizers of archaeal chromatin alongside classical histones.

biochemistry↗

bioRxiv2025

Modulation of archaeal hypernucleosome structure and stability by Mg2+

DNA-wrapping histone proteins play a central role in chromatin organization, gene expression and regulation in most eukaryotes and archaea. While the structure and function of eukaryotic histones are well-characterized, archaeal histones and their complexes with DNA require further scrutiny. Distinct from their eukaryotic counterparts, previously characterized canonical archaeal histones assemble on DNA into an endless superhelical nucleoprotein complex called a hypernucleosome. In this study, we explored whether hypernucleosome formation is a conserved feature of canonical archaeal histones. Moreover, to further elucidate how hypernucleosomes are regulated, we also explored how changes in the physico-chemical conditions, particularly the presence of Mg2+, influence the hypernucleosome. Using a combination of Tethered Particle Motion (TPM) and single-molecule force spectroscopy, we established that T. kodakarensis histones assemble into hypernucleosomes on DNA, similar to the M. fervidus histones HMfA and HMfB, the only canonical histones structurally characterized in previous studies. However, the effects of Mg2+ ions are distinct despite the histones high sequence- and structural similarity. We propose a model in which Mg2+ ions exert a generic effect on hypernucleosome compactness and stability due to electrostatic DNA shielding, with additional differential effects depending on histone identity.

molecular biology↗

bioRxiv2025

Cryo-EM reveals open and closed Asgard chromatin assemblies

Asgards are the closest archaeal relatives of eukaryotes, representing an important step in chromatin evolution. However, their chromatin organization has remained enigmatic until now. In this study, we present the first structures of Asgard chromatin assemblies formed by the Hodarchaeal histone HHoB. Our high-resolution cryo-EM structures reveal that this Asgard histone assembles into compact "closed" and into extended "open" hypernucleosomes. Thus the closed hypernucleosome conformation is conserved across archaeal lineages, while the open conformation resembles a eukaryotic H3-H4 octasome and likely represents an Asgard- specific innovation. Moreover, we show that Mg{superscript 2} ions influence Asgard chromatin conformation, suggesting a regulatory role. Overall, our study provides the first structure-based model of Asgard chromatin organization, expanding our understanding of chromatin architecture in evolutionary context.

biophysics↗

bioRxiv2025

DNA Wrapping by a Tetrameric Bacterial Histone

Histones are conserved DNA-packaging proteins found across all domains of life. In eukaryotes, canonical histones form octamers that wrap [~]147 base pairs of DNA into nucleosomes -- the fundamental units of chromatin. In archaea, histones form dimers that further multimerize into extended hypernucleosomes along DNA. Although bacteria were long thought to lack histones, recent studies have uncovered histone homologs in diverse bacterial lineages, many of which possess key DNA-binding features. We previously characterized HBb, a bacterial histone from Bdellovibrio bacteriovorus, which binds DNA as a dimer and induces bending. Here, we describe HLp from Leptospira perolatii, a representative of a distinct bacterial histone group. Crystallographic and biophysical analyses reveal that HLp forms stable tetramers. Like HBb, HLp binds DNA non-specifically; however, it adopts a different mode of interaction -- wrapping [~]60 base pairs of DNA around its tetrameric core. This wrapping mode, supported by molecular dynamics simulations and DNA-binding assays, promotes DNA compaction and alters its topology. When expressed heterologously in Escherichia coli, HLp reorganizes nucleoid morphology, consistent with a role in chromatin organization. These findings expand the known repertoire of histone-DNA interaction in bacteria and underscore the structural and functional diversity of histone-based genome organization across the tree of life.

biochemistry↗

bioRxiv2023

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. We show that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

molecular biology↗

bioRxiv2023

Novel histones and histone variant families in prokaryotes

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these new histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a new superfamily of histones, termed 3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.

bioinformatics↗

bioRxiv2021

Novel anti-repression mechanism of H-NS proteins by a phage "early protein".

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the early protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an "electrostatic zipper" between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their "half-open" conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

biochemistry↗

bioRxiv2021

The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

Bacterial chromosome structure is organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of Actinobacteria NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Deletion of gbn led to alterations in development and antibiotic production in Streptomyces coelicolor. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all such motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Transcriptomics analysis showed that Gbn binding generally leads to reduced gene expression. The DNA binding profiles were nearly identical between vegetative and aerial growth. Exceptions are SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which nearly exclusively bound during vegetative growth. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes. IMPORTANCEA large part of the chemical space of bioactive natural products is derived from Actinobacteria. Many of the biosynthetic gene clusters for these compounds are cryptic, in others words, they are expressed in nature but not in the laboratory. Understanding the global regulatory networks that control gene expression is key to the development of approaches to activate this biosynthetic potential. Chromosome structure has a major impact on the control of gene expression. In bacteria, the organization of chromosome structure is mediated by a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs), which play an important role in the control of gene expression, nucleoid structure and DNA repair. We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is a sporulation-specific protein that occurs only in the Actinobacteria and binds to GATC sequences, with a subtle but broad effect on global gene expression. The discovery of Gbn is a new step towards better understanding of how gene expression and chromosome structure is governed in antibiotic-producing streptomycetes.

molecular biology↗

bioRxiv2019

The B. subtilis Rok protein compacts and organizes DNA by bridging

Rok from Bacillus subtilis is an abundant DNA binding protein similar in function to H-NS-like proteins found in many proteobacteria. Rok binds across the genome with a preference for A/T rich DNA. Such DNA often contains genes of foreign origin that are silenced due to Rok binding. Rok also has been implied in global organization of the B. subtilis genome. However, how Rok binds to DNA and how it represses transcription is unclear. Also, it is unknown whether Rok-mediated gene repression can be induced or relieved following changes in physico-chemical conditions, as noted for H-NS-like proteins. Here we investigate the DNA binding properties of Rok and determine the effects of physico-chemical conditions on these properties. We demonstrate that Rok is a DNA bridging protein similar to H-NS like proteins from E. coli (H-NS), Pseudomonas sp. (MvaT) and Mycobacteria (Lsr2). Strikingly, unlike these proteins, the ability of Rok to bridge DNA is not affected by changes in physico-chemical conditions. Not being a direct sensor of such changes sets Rok apart from other H-NS like proteins. It implies the existence of other (protein-mediated) mechanisms to relieve Rok-mediated gene silencing in response to changes in environmental conditions.

molecular biology↗

bioRxiv2018

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA, modulate its conformation and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of C. difficile have been characterized to date.\n\nHere, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.\n\nThe present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.

microbiology↗

bioRxiv2016

Environment driven conformational changes modulate H-NS DNA bridging activity

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria, and is believed to be a crucial player in bacterial chromatin organization via its DNA bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the ability of H-NS to bridge DNA. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a \"closed\" and an \"open\", bridging competent, conformation driven by environmental cues and interaction partners.

biochemistry↗