Biology subjects

Dorman, C. J.

Publications and source records attributed to Dorman, C. J..

5 recordsLinked to original sources

bioRxiv2025

TORCphysics: A physical model of DNA-topology-controlled gene expression

DNA superhelicity and transcription are intimately related because changes to DNA topology can influence gene expression and vice versa. Information is transferred through the modulation of local DNA torsional stress, where the expression of one gene may influence the superhelical level of neighbouring genes, either promoting or repressing their expression. In this work, we introduce a one-dimensional physical model that simulates supercoiling-mediated regulation. This TORCphysics model takes as input a genome architecture represented either by a plasmid or by a linear DNA sequence with ends constrained under specific biological conditions, and computes the molecules output. Our findings demonstrate that the expression profiles of genes are directly influenced by the gene circuit design, including gene location, the positions of topological barriers, promoter sequences, and topoisomerase activity. The novelty that TORCphysics offers is versatility, where users can define distinct activity models for different types of proteins and protein binding sites. The aim of this research is to establish a flexible framework for developing physical simulations of gene circuits to deepen our comprehension of the intricate mechanisms involved in gene regulation.

biophysics↗

bioRxiv2025

Bacterial chromatin remodeling associated with transcription-induced domains at pathogenicity islands

The nucleoid-associated protein H-NS is a bacterial xenogeneic silencer responsible for preventing costly expression of genes acquired through horizontal gene transfer. H-NS silences several Salmonella Pathogenicity Islands (SPIs) essential for host infection. The stochastic expression of SPI-1 is required for invasion of host epithelial cells but complicates investigation of factors involved in SPI-1 chromatin structure and regulation. We performed functional genomics on sorted Salmonella populations expressing SPI-1 or not, to characterize how SPI-1 activation affects chromatin composition, DNA conformation, gene expression and SPI-1 subcellular localization. High H-NS occupancy co-exists with low spurious transcriptional activity and low RNA polymerase occupancy at silent SPIs. Upon SPI-1 activation, remodeling of H-NS occupancy defines a new chromatin landscape, which together with the master SPI-1 regulator HilD, facilitates transcription of SPI-1 genes. SPI-1 activation promotes formation of Transcription Induced Domains accompanied by repositioning SPI-1 close to the nucleoid periphery. We present a model for tightly regulated chromatin remodeling that minimizes the cost of pathogenicity island activation

microbiology↗

bioRxiv2022

The DNA-relaxation-dependent Off-to-On biasing of the type 1 fimbrial genetic switch requires the Fis nucleoid-associated protein.

The structural genes expressing type 1 fimbriae in Escherichia coli alternate between expressed (phase ON) and non-expressed (phase OFF) states due to inversion of the 314-bp fimS genetic switch. The FimB tyrosine integrase inverts fimS by site-specific recombination, alternately connecting and disconnecting the fim operon, encoding the fimbrial subunit protein and its associated secretion and adhesin factors, to and from its transcriptional promoter within fimS. Site-specific recombination by the FimB recombinase becomes biased towards phase ON as DNA supercoiling is relaxed, a condition that occurs when bacteria approach the stationary phase of the growth cycle. This effect can be mimicked in exponential phase cultures by inhibiting the negative DNA supercoiling activity of DNA gyrase. We report that this bias towards phase ON depends on the presence of the Fis nucleoid-associated protein. We mapped the Fis binding to a site within the invertible fimS switch by DNase I footprinting. Disruption of this binding site by base substitution mutagenesis abolishes both Fis binding and the ability of the mutated switch to sustain its phase ON bias when DNA is relaxed, even in bacteria that produce the Fis protein. In addition, the Fis binding site overlaps one of the sites used by the Lrp protein, a known directionality determinant of fimS inversion that also contributes to phase ON bias. The Fis-Lrp relationship at fimS is reminiscent of that between Fis and Xis when promoting DNA-relaxation-dependent excision of bacteriophage {lambda} from the E. coli chromosome. However, unlike the co-binding mechanism used by Fis and Xis at {lambda} attR, the Fis-Lrp relationship at fimS involves competitive binding. We discuss these findings in the context of the link between fimS inversion biasing and the physiological state of the bacterium.

microbiology↗

bioRxiv2021

The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

Integration host factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kilobase pairs apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1:1 stoichiometry, mass spectrometry revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the others gene. Mass spectrometry showed that in this rewired strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild type, had identical cell morphology, and was similar in competitive fitness. However, compared to the wild type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes. IMPACT STATEMENTIntegration Host Factor (IHF) is an abundant nucleoid-associated protein that organises DNA architecturally, influencing gene expression globally in Salmonella and other bacteria. IHF is composed of two related, non-identical, subunits, produced by genes that are 350 kilobase pairs apart. Each ihf gene has unique expression controls and is embedded in a complex genetic network that supports mRNA translation. Given that the subunits are thought to be required in a 1:1 ratio to form functional IHF, we were surprised by this physical and regulatory separation. We rewired the Salmonella genome so that each subunit was produced using the others regulatory signals and gene location. This revealed a high degree of tolerance to the effects of this rewiring. However, we discovered that bacterial motility was disrupted, as was the expression of virulence genes that have been acquired by horizontal gene transfer. Proteomic analysis using mass spectroscopy (MS) showed the extent of the alterations to cell composition. Our MS data also demonstrated that the subunits of IHF are not produced in a 1:1 ratio in either the wild type or the rewired strain. We discuss this finding in terms of the ability of each subunit to stabilise its partner. DATA SUMMARYO_LIWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309. C_LIO_LIData from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2). C_LIO_LIAll supporting data have been provided in the article or through supplementary data files. C_LI RepositoriesWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309. Data from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2).

microbiology↗

bioRxiv2020

Consequences of producing DNA gyrase from a synthetic gyrBA operon in Salmonella enterica serovar Typhimurium

DNA gyrase is an essential type II topoisomerase that is composed of two subunits, GyrA and GyrB and has an A2B2 structure. Although both subunits are required in equal proportions to form DNA gyrase, the gyrA and gyrB genes that encode them in Salmonella (and in many other bacteria) are at widely separated locations on the chromosome, are under separate transcriptional control and are present in different copy numbers in rapidly growing bacteria (gyrA is near the terminus of chromosome replication while gyrB is near the origin). We generated a synthetic gyrBA operon at the oriC-proximal location of gyrB to test the significance of the gyrase gene position for Salmonella physiology. Producing gyrase from an operon did not alter growth kinetics, cell morphology, competitive fitness index, or sensitivity to some gyrase-inhibiting antibiotics. However, the operon strain had altered DNA supercoiling set points, its SPI-2 virulence genes were expressed at a reduced level and its survival was reduced in macrophage. The gyrB gene could not be deleted from its oriC-proximal location, even in a gyrB merodiploid strain. We discuss the physiological significance of the different gyrA and gyrB gene arrangements found naturally in Salmonella and other bacteria.

microbiology↗