Biology subjects

Hardy, G. G.

Publications and source records attributed to Hardy, G. G..

4 recordsLinked to original sources

bioRxiv2025

Genome-wide DNA bridging by H-NS reshapes the stationary phase nucleoid and transcriptional landscape

Bacterial nucleoid-associated proteins (NAPs) structure the chromosome and regulate gene expression, but how these two functions are related is unclear. H-NS is a well-studied NAP that acts as a global gene silencer capable of bridging and looping DNA in vitro. Here, using high-throughput chromosome conformation capture assays, we show that H-NS mediates genome-wide long-range DNA looping in the stationary-phase nucleoid of Escherichia coli. Chromatin immunoprecipitation assays demonstrate that high levels of H-NS are present at the base of DNA loops. Super-resolution imaging and single-particle tracking show that H-NS binds more tightly in stationary phase and compacts the nucleoid mesh. Transcriptomic analyses indicate H-NS represses gene expression more strongly in the looped nucleoid and enables higher expression of genes outside of H-NS-bound regions. Overall, our study demonstrates that H-NS bridges distal DNA regions along the genome upon nutrient limitation, causing reduced nucleoid accessibility, stronger transcriptional repression, and a shifted transcriptional landscape.

molecular biology↗

bioRxiv2025

Dps binds and protects DNA in starved Escherichia coli with minimal effect on chromosome accessibility, dynamics and organisation

Dps is the most abundant nucleoid-associated protein in starved Escherichia coli with [~]180,000 copies per cell. Dps binds DNA and oxidises iron, facilitating survival in harsh environments. Dps-DNA complexes can form crystalline structures, leading to the proposed model that Dps reorganises the starved E. coli nucleoid into a compact liquid crystal, slowing chromosome dynamics and limiting access of other proteins to DNA. In this work, we directly tested this model using live-cell super-resolution microscopy and Hi-C analysis. We found that after 96 h of starvation, Dps compacts the nucleoid and increases short-range DNA-DNA interactions, but does not affect chromosome accessibility to large protein nanocages or small restriction enzymes. We also report that chromosome dynamics and organisation are primarily impacted by the bacterial growth phase; the effect of Dps is relatively minor. Our work clarifies the role of Dps in modulating nucleoid properties, and we propose an updated model for Dps-DNA interactions in which Dps binds, protects and compacts DNA largely without influencing chromosome access, dynamics and organisation. Additionally, this work provides a general framework for assessing the impact of nucleoid-associated proteins on key aspects of chromosome function in live cells. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/673347v3_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1385efforg.highwire.dtl.DTLVardef@1e4cb76org.highwire.dtl.DTLVardef@1e9e43dorg.highwire.dtl.DTLVardef@8768f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

HfaE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA to irreversibly adhere to surfaces. In C. crescentus, a freshwater Caulobacterales, the holdfast is anchored at the cell pole via the holdfast anchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD co-localize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not properly attached to the cell and shed into the medium. This phenotype is greater in a {Delta}hfaB than a double {Delta}hfaA hfaD mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similar to other anchor mutants. We showed that fluorescently-tagged HfaE co-localizes with holdfast, and HfaE forms an SDS-resistant high molecular weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein, and that HfaE functions to link holdfast material to the cell envelope. IMPORTANCEFor surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins and DNA. Species in the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces. In this study, we evaluate the role of a newly identified holdfast anchor protein HfaE in holdfast anchoring to the cell surface in two different Caulobacterales with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in Caulobacterales. Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.

microbiology↗

bioRxiv2021

Dual adhesive unipolar polysaccharides synthesized by overlapping biosynthetic pathways in Agrobacterium tumefaciens

Agrobacterium tumefaciens, is a member of the Alphaproteobacteria that pathogenizes plants, and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is comprised of two genetically, chemically and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each species. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific. Plain language summaryBacteria attach to a wide variety of surfaces including host tissues, via externalized structures described as adhesins. We define a large set of genes involved in synthesis of a complex unipolar adhesin comprised of two distinct polysaccharides, that is required for surface attachment by the plant-associated pathogen Agrobacterium tumefaciens.

microbiology↗