Biology subjects

Marsh, J. W.

Publications and source records attributed to Marsh, J. W..

7 recordsLinked to original sources

bioRxiv2026

ProFinder: Candidate constitutive promoter prediction across the bacterial tree for genetic tool development in non-model species

Reliable constitutive promoters for non-model bacteria are lacking, resulting in a bottleneck for genetic tool development. Existing computational promoter predictors are trained on model Gammaproteobacteria and degrade on phylogenetically distant or compositionally atypical genomes. Here we present ProFinder, a computational method that integrates three biologically informed criteria for promoter prediction: a conserved 98-gene constitutive marker panel, an empirically derived intergenic search window, and a 72-motif {sigma}70 motif catalogue built from diverse lineages. From an input bacterial assembly, ProFinder returns a ranked shortlist of putative constitutive promoters. It matches the precision of full-genome classifiers and is more robust to GC extremes. Applied to 56,742 bacterial genomes spanning 149 phyla, ProFinder returned candidate promoters for 99.76%, delivering a pan-bacterial catalogue of pre-computed constitutive promoter parts to accelerate chassis development across the bacterial tree. ProFinder can be accessed at: https://plabase.cs.uni-tuebingen.de/profinder/.

microbiology↗

bioRxiv2025

Genetic modification of Clostridium kluyveri for heterologous n-butanol and n-hexanol production

The mesophilic microbe Clostridium kluyveri serves as the most commonly used model microbe to elucidate the physiology and biochemistry of ethanol-based chain elongation via reverse {beta}-oxidation. In this pathway, ethanol and acetate are converted into short- and medium-chain carboxylates. However, to date, no genetic system has been published in a peer-reviewed publication. Here, we report the development of versatile genetic tools for C. kluyveri, utilizing the pMTL Clostridia shuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system of C. kluyveri as a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern of C. kluyveri, we performed in-vivo methylation of the shuttle vector plasmid by expressing the methyltransferase in Escherichia coli, followed by DNA transfer via conjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH and NADPH-dependent alcohol dehydrogenases from Clostridium acetobutylicum. The expression of these genes was controlled by the Pthl promoter, which is commonly used in Clostridia, and the PadhE2 promoter, leading to n-butanol and n-hexanol production of the mutant strains. This genetic system for C. kluyveri will not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general. IMPORTANCEMedium-chain carboxylates are required in various everyday products, including cosmetics, pharmaceuticals, and fragrances, and show a natural antimicrobial property. Further, they represent food additives and serve as chemical building blocks for several other compounds. Traditionally, these carboxylates are produced from fossil resources, contributing to increased greenhouse gas emissions. Alternatively, they are derived from animal- or plant-based fat (e.g., coconut oil), which competes with agricultural land that is needed for food production. However, microbial chain elongation, which is a biotechnological approach relying on microbes, such as Clostridium kluyveri, is sustainable and a promising alternative to the conventional production of medium-chain carboxylates. Notably, it enables the use of industrial waste streams (e.g., off-gases, carbohydrate-rich industrial waste) as substrates, making the process more environmentally friendly. By applying our genetic system for C. kluyveri, a better understanding of microbial chain elongation can be achieved, and potentially even an extension of its product portfolio.

microbiology↗

bioRxiv2021

Genomic diversity of hospital-acquired infections revealed through prospective whole genome sequencing-based surveillance

Healthcare-associated infections (HAIs) cause mortality, morbidity, and waste of healthcare resources. HAIs are also an important driver of antimicrobial resistance, which is increasing around the world. Beginning in November 2016, we instituted an initiative to detect outbreaks of HAI using prospective whole genome sequencing-based surveillance of bacterial pathogens collected from hospitalized patients. Here we describe the biodiversity of bacteria sampled from hospitalized patients at a single center, as revealed through systematic analysis of their genomes. We sequenced the genomes of 3,004 bacterial isolates from hospitalized patients collected over a 25-month period. We identified bacteria belonging to 97 distinct species, which were distributed among 14 species groups. Within these groups, isolates could be distinguished from one another by both average nucleotide identity (ANI) and principal component analysis of accessory genes (PCA-A). Genetic distances between isolates and rates of evolution varied between different species, which has implications for the selection of distance cut-offs for outbreak analysis. Antimicrobial resistance genes and the sharing of mobile genetic elements between different species were frequently observed. Overall, this study describes the population structure of pathogens circulating in a single healthcare setting, and shows how investigating microbial population dynamics can inform genomic epidemiology studies. ImportanceHospitalized patients are at increased risk of becoming infected with antibiotic-resistant organisms. We used whole-genome sequencing to survey and compare over 3,000 bacterial isolates collected from hospitalized patients at a large medical center over a two-year period. We identified nearly 100 different bacterial species, suggesting that patients can be infected with a wide variety of different organisms. When we examined how genetic relatedness differed between species, we found that different species are likely evolving at different rates within our hospital. This is significant because the identification of bacterial outbreaks in the hospital currently relies on genetic similarity cut-offs, which are often applied uniformly across organisms. Finally, we found that antibiotic resistance genes and mobile genetic elements were abundant among the bacterial isolates we sampled. Overall, this study provides an in-depth view of the genomic diversity and evolution of bacteria sampled from hospitalized patients, as well as genetic similarity estimates that can inform hospital outbreak detection and prevention efforts.

microbiology↗

bioRxiv2019

Comprehensive analysis of horizontal gene transfer among multidrug-resistant bacterial pathogens in a single hospital

Multidrug-resistant bacterial pathogens pose a serious public health threat, especially in hospital settings. Horizontal gene transfer (HGT) of mobile genetic elements (MGEs) contributes to this threat by facilitating the rapid spread of genes conferring antibiotic resistance, enhanced virulence, and environmental persistence between nosocomial pathogens. Despite recent advances in microbial genomics, studies of HGT in hospital settings remain limited in scope. The objective of this study was to identify and track the movement of MGEs within a single hospital system using unbiased methods. We screened the genomes of 2,173 bacterial isolates from healthcare-associated infections collected over an 18-month time period to identify nucleotide regions that were identical in the genomes of bacteria belonging to distinct genera. These putative MGEs were found in 196 isolates belonging to 11 different genera; they grouped into 51 clusters of related elements, and they were most often shared between related genera. To resolve the genomic locations of the most prevalent MGEs, we performed long-read sequencing on a subset of representative isolates and generated highly contiguous, hybrid-assembled genomes. Many of these genomes contained plasmids and chromosomal elements encoding one or more of the MGEs we identified, which were often arranged in a mosaic fashion. We then tracked the appearance of ten MGE-bearing plasmids in all 2,173 genomes, and found evidence supporting the transfer of plasmids between patients independent from bacterial transmission. Finally, we identified two instances of likely plasmid transfer across genera within individual patients. In one instance, the plasmid appeared to have subsequently transferred to a second patient. By surveying a large number of bacterial genomes sampled from infections at a single hospital in a systematic and unbiased manner, we were able to track the independent transfer of MGEs over time. This work expands our understanding of HGT in healthcare settings, and can inform efforts to limit the spread of drug-resistant pathogens in hospitals.

microbiology↗

bioRxiv2019

Early transcriptional landscapes of Chlamydia trachomatis-infected epithelial cells at single cell resolution

Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. C. trachomatis causes trachoma (infectious blindness) in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility of single cell approaches to identify early host cell biomarkers of chlamydial infection. 264 time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-hour cells from 6- and 12-hours. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. Changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.

systems biology↗

bioRxiv2019

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues

systems biology↗

bioRxiv2017

Dual RNA sequencing (dRNA-Seq) of bacteria and their host cells

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA-Seq technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. We pioneered dRNA-Seq to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory and bioinformatics protocol for dRNA-seq that is readily adaptable to any host-bacteria system of interest.

genomics↗