Biology subjects

Horne, J.

Publications and source records attributed to Horne, J..

4 recordsLinked to original sources

bioRxiv2025

A lipoprotein partner for the Escherichia coli outer membrane protein TolC

The outer-membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologs bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular and modification-membrane interactions seen in TolC homologs. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. Although the association occurs spontaneously both in vitro and in vivo, the YbjP-TolC interaction is not required for efflux activity under standard laboratory conditions. YbjP may contribute stabilising the orientation and distribution of TolC in the outer membrane as well as the expression of transporters for tryptophan and cyclic peptide toxins.

molecular biology↗

bioRxiv2024

The phage protein paratox is a multifunctional metabolic regulator of Streptococcus

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a commensal bacteria and human pathogen. Central to GAS pathogenesis is the presence of prophage encoded virulence genes. The conserved phage gene for the protein paratox (Prx) is genetically linked to virulence genes, but the reason for this linkage is unknown. Prx inhibits GAS quorum sensing and natural competence by binding the transcription factor ComR. However, inhibiting ComR does not explain the virulence gene linkage. To address this, we took a mass-spectrometry approach to search for other Prx interaction partners. The data demonstrates that Prx binds numerous DNA binding proteins and transcriptional regulators. We show binding of Prx in vitro with the GAS protein EndoS1 (SpyM3_0890) and the phage protein JM3 (SpyM3_1246). An EndoS1:Prx complex X-ray crystal structure reveals that EndoS1 and ComR possess a conserved Prx binding helix. Computational modelling predicts that the Prx binding helix is present in several, but not all, binding partners. Namely, JM3 lacks the Prx binding helix. As Prx is conformationally dynamic, this suggests partner-dependent binding modes. Overall, Prx acts as a metabolic regulator of GAS to maintain the phage genome. As such, Prx maybe a direct contributor to the pathogenic conversion of GAS.

biochemistry↗

bioRxiv2019

A robust metatranscriptomic technology for population-scale studies of diet, gut microbiome, and human health

A functional readout of the gut microbiome is necessary to enable precise control of the gut microbiomes functions, which support human health and prevent or minimize a wide range of chronic diseases. Stool metatranscriptomic analysis offers a comprehensive functional view of the gut microbiome, but despite its usefulness, it has rarely been used in clinical studies due to its complexity, cost, and bioinformatic challenges. This method has also received criticism due to potential intra-sample variability, rapid changes, and RNA degradation. Here, we describe a robust and automated stool metatranscriptomic method, called Viomega, which was specifically developed for population-scale studies. Viomega includes sample collection, ambient temperature sample preservation, total RNA extraction, physical removal of ribosomal RNAs (rRNAs), preparation of directional Illumina libraries, Illumina sequencing, taxonomic classification based on a database of >110,000 microbial genomes, and quantitative microbial gene expression analysis using a database of ~100 million microbial genes. We applied this method to 10,000 human stool samples, and performed several small-scale studies to demonstrate sample stability and consistency. In summary, Viomega is an inexpensive, high throughput, automated, and accurate sample-to-result stool metatranscriptomic technology platform for large-scale studies and a wide range of applications.

systems biology↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗