Biology subjects

Jaschke, P. R.

Publications and source records attributed to Jaschke, P. R..

15 recordsLinked to original sources

bioRxiv2026

Host background shapes the portability of a non-canonical translation initiation system across Escherichia coli strains

Translation initiation has become an attractive target for engineering orthogonal translation systems, yet the extent to which these systems retain functionality across distinct host backgrounds remains poorly defined. In bacteria, start codon recognition depends on pairing between the initiator tRNA anticodon and a suitable start codon within the appropriate distance from the Shine-Dalgarno sequence. These sequence-specific interactions enable translation initiation to be reprogrammed through anticodon engineering. What is currently missing is an understanding of how anticodon mutants of initiator tRNAs function across different bacterial strains. Here, we systematically evaluated the portability of a library of twelve i-tRNA anticodon mutants paired with their complementary non-canonical start codons. Most i-tRNA-start codon pairs supported detectable translation initiation across multiple strains, demonstrating broad functional portability. However, initiation efficiency, absolute system output, and fitness effects varied substantially between strains. Comparative genomic analyses revealed host-specific gene differences broadly, and endogenous tRNA gene sequence and copy number specifically, was associated with this variability. While most i-tRNA variants were well tolerated, a subset produced strain-dependent growth defects that primarily affected growth rate rather than final culture density. Together, these findings show that translation initiation efficacy of engineered i-tRNAs is partially strain-dependent and that host background must be considered a key design variable when deploying these translation systems. Looking forward, this study provides a framework for host-aware selection of microbial chassis for orthogonal translation applications in synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/719103v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@9c75deorg.highwire.dtl.DTLVardef@1fe704borg.highwire.dtl.DTLVardef@17e7808org.highwire.dtl.DTLVardef@1fa6fa1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Defining bottlenecks and physiological impact of an orthogonal translation initiation system

Reengineering translation initiation provides a powerful route to develop new translation systems that enable precise control of protein synthesis. While many engineered translation systems show promise, their orthogonality and impact on host physiology is largely uncharacterized, limiting broader application. Here, we develop an initiator-tRNA with an AAC anticodon mutation (i-tRNA-AAC), enabling translation initiation at a GUU start codon. Using fluorescence assays, proteomics, and tRNA sequencing, we assess the i-tRNA-AAC initiation orthogonality and effects on the host, guiding the optimization of its translational efficiency. We find the i-tRNA-AAC mutant initiates translation exclusively from its GUU start codon and is improved by overexpression of valyl-tRNA synthetase and methionyl-tRNA formyltransferase. However, this intervention perturbs aminoacylation and base modifications of endogenous tRNAs along with proteome-wide changes likely due to increased valine demand. Our findings demonstrate how an orthogonal translation initiation system reshapes host physiology and reveals the adaptive responses that accompany translational reprogramming.

synthetic biology↗

bioRxiv2025

Improved in silico and in vitro methods for E. coli LPS outer core typing

The Gram-negative bacterial envelope comprises the outer membrane, periplasmic space with the peptidoglycan layer, and inner (cytoplasmic) membrane. A lipopolysaccharide (LPS) layer linked to the outer membrane is essential for survival in most species of bacteria, primarily by providing structural stability and regulating selective chemical permeability. These functions make the LPS layer a key pathogenicity determinant, protecting bacteria from host defences. At the same time, it serves as a common receptor for multiple bacteriophage orders, making it a crucial point of bacterial vulnerability. LPS outer core typing is traditionally performed using immunoblotting and PCR. With the increasing availability of sequenced genomes, PCR has emerged as the primary method for typing. This study presents a set of nine oligonucleotides designed for typing the five LPS outer core structures of Escherichia coli: R1, R2, R3, R4, and K-12, both in vitro and in silico. Our method was able to successfully type 99% of strains within a comprehensive dataset of 4549 complete genomes. Using these new methods, we identify previously unreported hybrid LPS structures involving R2 and K-12 outer core types and establish associations between LPS outer core types and E. coli phylogenetic groups, pathogenicity, O-antigen polysaccharides, and capsule types. We also introduce LPSTyper, a Python-based command-line tool that enables rapid and precise LPS outer core typing in E. coli genome sequences. Together, the expanded dataset of LPS outer core types and enhanced laboratory and computational tools for their detection provide new insights into LPS structural evolution and its role in E. coli ecology and pathogenicity. We anticipate these tools will improve the efficiency of future research and diagnostics of this important bacterial envelope structure. Impact StatementBacterial lipopolysaccharide (LPS) layer types are important for determining both pathogenicity potential and phage susceptibility. This study presents a robust and scalable framework for precise typing of Escherichia coli lipopolysaccharide (LPS) outer core types, enabled by the development of nine novel oligos targeting the waa locus. Using the LPSTyper tool for in silico analysis and a simple PCR-based method for in vitro validation, this approach allows rapid and accurate typing across thousands of genomes, supporting large-scale monitoring of LPS diversity in both commensal and pathogenic E. coli populations. Our findings reveal strong associations between LPS outer core types, O-antigens, capsules, phylogenetic groups, and pathotypes, highlighting the critical role of LPS structure in virulence, immune evasion, and environmental adaptation. The discovery of hybrid and novel waa locus arrangements further underscores the evolutionary plasticity of LPS biosynthesis. Overall, this work provides essential tools and insights for genomic surveillance and deepens our understanding of E. coli surface structure variability central to host-pathogen interactions and antimicrobial resistance. Data SummaryThe LPSTyper source code is freely available on GitHub under the Apache 2.0 licence: https://github.com/ellinium/LPSTyper. Sequencing data for all genomes used in this study are accessible via the NCBI database under BioProject accession number PRJNA1243169. Individual genome accession numbers are listed in Table 2. All supporting data are provided in Supplementary Data Files 1 and 2. O_TBL View this table: org.highwire.dtl.DTLVardef@951af3org.highwire.dtl.DTLVardef@11c790eorg.highwire.dtl.DTLVardef@4fb5e3org.highwire.dtl.DTLVardef@12e87caorg.highwire.dtl.DTLVardef@1d4fc1a_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONReference E. coli strains used in in vitro PCR. C_TABLECAPTION C_TBL

microbiology↗

bioRxiv2024

Designing a simple and efficient phage biocontainment system using the amber suppressor

Multidrug-resistant infections are becoming increasingly prevalent worldwide. One of the fastest emerging alternative and adjuvant therapies being proposed is phage therapy. Naturally-isolated phages are used in the vast majority of phage therapy treatments today. Engineered phages are being developed to enhance the effectiveness of phage therapy, but concerns over their potential escape remains a salient issue. To address this problem, we designed a biocontained phage system based on conditional replication using amber stop codon suppression. This system can be easily installed on any natural phage with a known genome sequence. To test the system, we mutated the start codons of three essential capsid genes in the phage {phi}X174 to the amber stop codon (TAG). These phages were able to efficiently infect host cells expressing the amber initiator tRNA, which suppresses the amber stop codon and initiates translation at TAG stop codons. The amber phage mutants were also able to successfully infect host cells and reduce their population on solid agar and liquid culture but could not produce infectious particles in the absence of the amber initiator tRNA or complementing capsid gene. We did not detect any growth-inhibiting effects on E. coli strains known to lack a receptor for {phi}X174, and we show that engineered phages have a limited propensity for reversion. The approach outlined here may be useful to control engineered phage replication in both the lab and clinic.

microbiology↗

bioRxiv2024

Discovery and characterisation of new phage targeting uropathogenic Escherichia coli

Antimicrobial resistance (AMR) is increasing at an escalating rate with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are particularly prone to becoming recurrent and antibiotic resistant. The aim of this study was to discover and characterise new bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), which is the leading cause of UTIs. Six phages from the Autographiviridae family and Guernseyvirinae sub-family were isolated from wastewater and sequenced. The length of the isolated phage genomes was between 39,471 bp and 45,233 bp, with a GC content between 45.0% and 51.0%, and 57 to 84 predicted coding sequences (CDS) per genome. These phages were found to infect between 25 - 75% of the twelve UPEC strains tested. Using sequence comparison and predicted structural alignments, we show a similarity between the C-terminal domain of the tail fiber proteins of two phage that correlates with their host range. In vitro characterisation of phage cocktails against a single bacterial strain did not perform better than the best-performing phage, but did show synergistic improvement against a mixed UPEC strain population. Lastly, we measured the effectiveness of treatment with phage with different lytic kinetics in a sequential treatment and found it was improved over single phage treatment.

microbiology↗

bioRxiv2022

IbpAB small heat shock proteins are not host factors for bacteriophage {varphi}X174 replication

Bacteriophages exploit host proteins for successful infection. Small heat shock proteins are a universally conserved family of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Two small heat shock proteins, IbpA and IbpB, are a class of holding modulators or "holdases", which bind partially folded proteins and await ATP-driven folding chaperones for refolding. Bacteriophage {phi}X174 is a small, icosahedral, and non-tailed virus belonging to the Microviridae. During {phi}X174 infection of Escherichia coli C122, IbpA and IbpB were previously found to be the most highly upregulated host proteins, with expression levels comparable to {phi}X174 proteins. In this work, to understand the role of IbpA and IbpB during {phi}X174 infection, we used a hybrid approach of CRISPR interference and genomic knockouts to disrupt the ibpA and ibpB genes. We show that these two proteins do not appear to be necessary for efficient {phi}X174 replication, and moreover, their absence has no effect on {phi}X174 fecundity. ImportanceThe small heat shock proteins (sHsps) are universally conserved family of stress-induced molecular chaperones that prevent irreversible protein aggregation. In E. coli, the IbpA/B sHsps work together, and separately, to bind partially folded proteins and await ATP-driven folding chaperones for refolding. These proteins are highly upregulated during protein overexpression and bacteriophage infection, but their collective role in bacteriophage infection has not been investigated. Here, we show that the ibpA/B genes are dispensable for bacteriophage {phi}X174 infection, and are likely not essential host factors despite their abundance during diverse phage infections. Instead, this work points towards their role as cell wall integrity sensors, similar to the phage shock protein system, in addition to their canonical role as holdases of cytoplasmic protein.

microbiology↗

bioRxiv2022

Creating De Novo Overlapped Genes

Future applications of synthetic biology will rely on deploying engineered cells outside of lab environments for long periods of time. Currently, a significant roadblock to this application is the potential for deactivating mutations in engineered genes. A recently developed method to protect engineered coding sequences from mutation is called Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS). In this chapter we provide a workflow for utilising CAMEOS to create synthetic overlaps between two genes, one essential (infA) and one non-essential (aroB), to protect the non-essential gene from mutation and loss of protein function. In this workflow we detail the methods to collect large numbers of related protein sequences, produce multiple sequence alignments (MSAs), use the MSAs to generate Hidden Markov Models and Markov Random Field models, and finally generate a library of overlapping coding sequences through CAMEOS scripts. To assist practitioners with basic coding skills to try out the CAMEOS method, we have created a virtual machine containing all the required packages already installed, that can be downloaded and run locally.

synthetic biology↗

bioRxiv2021

A Codon Constrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters

Future applications of synthetic biology will require refactored genetic sequences devoid of internal regulatory elements within coding sequences. These regulatory elements include cryptic and intragenic promoters which may constitute up to a third of predicted Escherichia coli promoters. Promoter activity is dependent on the structural interaction of core bases with a {sigma} factor. Rational engineering can be used to alter key promoter element nucleotides interacting with {sigma} factors and eliminate downstream transcriptional activity. In this paper, we present COdon Restrained Promoter SilEncing (CORPSE), a system for removing intragenic promoters. CORPSE exploits the DNA-{sigma} factor structural relationship to disrupt {sigma}70 promoters embedded within gene coding sequences, with a minimum of synonymous codon changes. Additionally, we present an inverted CORPSE system, iCORPSE, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.

synthetic biology↗

bioRxiv2021

Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo

Biological engineers seek to have better control and a more complete understanding of the process of translation initiation within cells so that they may produce proteins more efficiently, as well as to create orthogonal translation systems. Previously, initiator tRNA variants have been created that initiate translation from non-AUG start codons, but their orthogonality has never been measured and the detailed characteristics of proteins produced from them have not been well defined. In this study we created an initiator tRNA mutant with anticodon altered to AAC to be complementary to GUU start codons. We deploy this i-tRNA(AAC) into E. coli cells and measure translation initiation efficiency against all possible start codons. Using parallel reaction monitoring targeted mass spectrometry we identify the N-terminal amino acids of i-tRNA(AAC)-initiated reporter proteins and show these proteins have altered stability within cells. We also use structural modeling of the peptide deformylase enzyme interaction with position 1 valine peptides to interrogate a potential mechanism for accumulation of formylated-valine proteins observed by mass spectrometry. Our results demonstrate that mutant initiator tRNAs have potential to initiate translation more orthogonally than the native initiator tRNA but their interactions with cellular formyltransferases and peptide deformylases can be inefficient because of the amino acid they are charged with. Additionally, engineered initiator tRNAs may enable tuning of in vivo protein stability through initiation with non-methionine amino acids that alter their interaction with cellular proteases.

synthetic biology↗

bioRxiv2021

Plaque Size Tool: an automated plaque analysis tool for simplifying and standardising bacteriophage plaque morphology measurements

Bacteriophage plaque size measurement is essential for phage characterisation, but their manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bulls-eye morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows.

microbiology↗

bioRxiv2020

Proteomic and transcriptomic analysis of Microviridae {varphi}X174 infection reveals broad up-regulation of host membrane damage and heat shock responses

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage {varphi}X174 is endemic to the human gut and has been studied for over seventy years but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome we have measured host Escherichia coli C proteomic and transcriptomic response to {varphi}X174 infection. We used mass spectrometry and RNA-seq to identify and quantify all 11 {varphi}X174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in {varphi}X174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the {varphi}X174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat-shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model phage. IMPORTANCEA major part of the healthy human gut microbiome are the Microviridae bacteriophage, exemplified by the model {varphi}X174 phage. Although much has been learned from studying {varphi}X174 over the last half century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the {varphi}X174 infection cycle, and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

microbiology↗

bioRxiv2020

Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction

ABSTRACTSequence overlap between two genes is common across all genomes, with viruses having high proportions of these gene overlaps. The biological function and fitness effects of gene overlaps are not fully understood, and their effects on gene cluster and genome-level refactoring are unknown. The bacteriophage ϕX174 genome has ∼26% of nucleotides involved in encoding more than one gene. In this study we use an engineered ϕX174 phage containing a genome with all gene overlaps removed, to show that gene overlap is critical to maintaining optimal viral fecundity. Through detailed phenotypic measurements we reveal that genome modularization in ϕX174 causes virion replication, stability, and attachment deficiencies. Quantitation of the complete phage proteome across an infection cycle reveals almost half the proteins display abnormal expression patterns. Taken together, we have for the first time comprehensively demonstrated that gene modularization severely perturbs the coordinated functioning of a bacteriophage replication cycle. This work highlights the biological importance of gene overlap in natural genomes and that reducing gene overlap disruption should be an integral part of future genome engineering projects.View Full Text

synthetic biology↗

bioRxiv2020

A high-resolution map of bacteriophage {Phi}X174 transcription

Bacteriophage oX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiome, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from oX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical oX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae family, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.

genomics↗

bioRxiv2019

Measuring amber initiator tRNA orthogonality in a genomically recoded organism

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.{Delta}A.exp lacking all UAG stop codons was created, freeing this amber stop codon for other purposes. An engineered amber initiator [Formula] that activates translation at UAG codons is available, but little is known about this tRNAs orthogonality. Here, we combine for the first time the amber initiator [Formula]in C321.{Delta}A.exp and measure its cellular effects. We found that the [Formula]expression resulted in a nearly 200Yfold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosomeYassociated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for offYtarget translation initiation. In contrast to previous work, we show that UAGYinitiated proteins carry NYterminal methionine exclusively. Together, our results identify beneficial features of using the amber initiator [Formula]to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

synthetic biology↗

bioRxiv2018

Definitive demonstration by synthesis of genome annotation completeness

Bacteriophage oX174 was the first DNA genome to be sequenced. The genome is well studied by classical methods and is known to encode 11 essential genes. At least 23 closely-related Bullavirinae genome sequences are now available. We identified 315 potential open reading frames (ORFs) within the genome via bioinformatic analysis, and a subset of 82 highly-conserved ORFs that have no known gene products or functions. Using genome scale design and synthesis we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved-but-cryptic ORFs. The resulting genome is not viable, as expected. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic-ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ~50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation modulating translation of gene H, a known essential gene, that fully suppressed the fitness defect. Taken together, we conclude that the annotation of ORFs for the oX174 genome is formally complete. Sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

synthetic biology↗