Biology subjects

Hospenthal, M. K.

Publications and source records attributed to Hospenthal, M. K..

4 recordsLinked to original sources

bioRxiv2026

Structural and biochemical insights reveal substrate-modulated nuclease activity of ComEC during DNA processing

Natural transformation enables bacteria to internalise extracellular DNA, driving adaptation and the spread of antibiotic resistance. The membrane protein ComEC mediates translocation of single-stranded DNA across the cytoplasmic membrane while degrading the complementary strand, yet the structural basis of its activity remains incompletely defined. Here, we report a cryo-electron microscopy structure of full-length ComEC from Neomoorella carbonis in a pre-translocation state, revealing a three-domain architecture and a conserved transmembrane channel captured in a closed conformation. Structural analysis indicates that conformational rearrangements of channel-lining helices are required to accommodate single-stranded DNA. Biochemical assays show that, relative to the isolated {beta}-lactamase-like domain, full-length ComEC degrades DNA more efficiently. Importantly, coating of the DNA by the periplasmic DNA receptor ComEA suppresses endonucleolytic cleavage, thereby modulating nuclease activity. Together, these findings provide the first characterisation of the nuclease activity of full-length ComEC and show how ComEA-mediated protection of the substrate directs ComECs nuclease activity to ensure high fidelity during the natural transformation process.

biochemistry↗

bioRxiv2025

Molecular interplay between ComEC domains leads to efficient DNA translocation during natural transformation

Naturally competent bacteria can take up and incorporate free DNA from their environment using complex machinery that is endogenously encoded. This process is called natural transformation and is a key mechanism in the spread of antibiotic resistance amongst bacteria, including many human pathogens. All competent bacteria require ComEC to transport the transforming DNA across the cytoplasmic membrane. In addition to the transmembrane competence domain predicted to form the DNA channel, most ComEC orthologues additionally contain an oligonucleotide binding (OB) domain and {beta}-lactamase-like domain. Here, we provide in-depth characterisation of the nuclease activity of the {beta}-lactamase-like domain and the DNA binding activity of the OB domain, and present high-resolution structures of both domains. We show that the in vitro nuclease activity of the {beta}-lactamase-like domain is enhanced when the OB domain is encoded on the same polypeptide chain. Additionally, we identify a pin loop within the {beta}-lactamase-like domain responsible for melting the DNA duplex prior to cleavage of the non-translocating strand, and a DNA channel lined with aromatic residues that guide the uncleaved translocating strand through ComEC. On the basis of our biochemical, structural and functional characterisation, we provide a mechanistic model for how ComEC achieves the simultaneous tasks of DNA degradation and translocation, central to the natural transformation process.

microbiology↗

bioRxiv2021

The Molecular Basis of FimT-mediated DNA Uptake during Bacterial Natural Transformation

Naturally competent bacteria encode sophisticated protein machineries for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. In this study, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We used NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimTs role in this process. FimT directly interacts with DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in FimTs conformationally flexible C-terminal tail. We also show that FimT orthologues from other {gamma}-Proteobacteria share the ability to bind to DNA. Our functional characterisation and comprehensive bioinformatic analysis of FimT, suggest that it plays an important role for DNA uptake in a wide range of competent species.

microbiology↗

bioRxiv2017

The Cryo-Electron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod

Chaperone-usher pili are long, polymeric protein fibres displayed on the surface of many bacterial pathogens. These critical virulence factors allow bacteria to specifically attach to host cells during infection. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during UPECs colonisation of the urinary tract, mediating bacterial attachment to the bladder and kidney, respectively. Also, their biomechanical properties that allow them to reversibly uncoil in response to flow-induced forces are critical for UPECs ability to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2 [A] resolution cryo-electron microscopy (cryo-EM) structure of the type 1 pilus rod, which together with the previous structure of the P pilus rod, enables us to understand the remarkable \"spring-like\" properties of chaperone-usher pili in more detail.

molecular biology↗