Biology subjects

Goodall, E. C. A.

Publications and source records attributed to Goodall, E. C. A..

7 recordsLinked to original sources

bioRxiv2026

Transposon mutagenesis uncovers the genetic landscape of streptomycin susceptibility and implicates SbmA in aminoglycoside uptake in Escherichia coli

Aminoglycosides are critical antibiotics with partially elucidated mechanisms of uptake and action in Gram-negative bacteria. Importantly, although energy-dependent uptake across the inner membrane has been well-established, the specific molecular mechanisms involved have not been definitively identified. To deepen understanding of genetic factors influencing susceptibility and resistance to streptomycin, we applied transposon insertion sequencing in Escherichia coli K-12. This approach identified both known and novel genes whose disruption increased susceptibility, including those involved in respiration, protein export, cell division, and uncharacterised functions. Notably, voltage-sensitive membrane dye-based assays revealed that many susceptible mutants did not display inner membrane hyperpolarisation as often assumed. Conversely, disruption of certain genes, such as the inner membrane antimicrobial peptide transporter sbmA, conferred low-level resistance, with sbmA overexpression increasing streptomycin sensitivity, suggesting its role in aminoglycoside uptake. These findings refine the model of aminoglycoside interaction with various pathways and highlight potential targets for adjuvant therapies to combat antimicrobial resistance.

microbiology↗

bioRxiv2026

Vi-TIS: a transposon insertion sequencing framework for quantitative discrimination of viable and non viable mutants

Transposon-insertion sequencing (TIS) is a high-throughput approach that uses randomly generated transposon mutant libraries to assess bacterial gene essentiality and their contribution to fitness under defined conditions. The method integrates classical transposon mutagenesis with next-generation sequencing. However, conventional TIS workflows generally require at least one active growth step to enrich for replicating or fit mutants. The outcome of these experiments can be biased by the recovery of non-replicating cells or by the loss of slow-growing mutants. To overcome this limitation, we modified the traditional TIS workflow by incorporating propidium monoazide (PMA). PMA is membrane-impermeant and excluded from viable cells but can enter dead cells when the membrane has lost its integrity. PMA covalently crosslinks to DNA upon exposure to UV light. Such binding inhibits PCR amplification, an essential step in TIS library preparation. This property enables selective exclusion of DNA originating from dead cells and reduces the signal to noise ratio in TIS experiments. We refer to this modified approach as Viability-TIS (Vi-TIS). Applying Vi-TIS to our ultra-dense Escherichia coli K-12 BW25113 TIS library enhanced the accuracy of essential gene identification. We further exposed the library to subinhibitory concentrations of carbenicillin and found that Vi-TIS produced results consistent with those obtained using culture-based methodologies. In addition, Vi-TIS revealed previously unrecognized carbenicillin-susceptible mutants. Finally, we demonstrate that the mutations are linked to perturbations in peptidoglycan biosynthesis.

microbiology↗

bioRxiv2024

Identification of new genes on a whole genome scale using saturated reporter transposon mutagenesis

Small or overlapping genes are prevalent across all domains of life but are often overlooked for annotation and function because of challenges in their detection. The advent of high-density mutagenesis and data-mining studies suggest the existence of further coding potential within bacterial genomes. To overcome limitations in existing protein detection methods, we applied a genetics-based approach. We combined transposon insertion sequencing with a translation reporter to identify translated open reading frames throughout the genome at scale, independent of genome annotation. We applied our method to the well-characterised species Escherichia coli and identified [~]200 putative novel protein coding sequences (CDS). These are mostly short CDSs (<50 amino acids) and in some cases highly conserved. We validated the expression of selected CDSs demonstrating the utility of this approach. Despite the extensive study of E. coli, this method revealed proteins that have not been described previously, including proteins that are conserved and neighbour functionally important genes, suggesting significant functional roles of these small proteins. We present this as a complementary method to whole cell proteomics and ribosome trapping for condition-dependent identification of protein CDSs, and as a high-throughput method for testing conditional gene expression. We anticipate this technique will be a starting point for future high-throughput genetics investigations to determine the existence of unannotated genes in multiple bacterial species.

microbiology↗

bioRxiv2023

Bam complex associated proteins in Escherichia coli are functionally linked to peptidoglycan biosynthesis, membrane fluidity and DNA replication

Biogenesis of the bacterial outer membrane is key to bacterial survival and antibiotic resistance. Central to this is the {beta}-barrel assembly machine (Bam) complex and its associated chaperones, which are responsible for transport, folding and insertion of outer membrane proteins (OMPs). The Escherichia coli Bam complex is composed of two essential subunits, BamA and BamD, and three non-essential accessory lipoproteins, BamB, BamC and BamE. Optimal Bam function is further dependent on the non-essential periplasmic chaperones DegP, Skp and SurA. Despite intensive study, the specific function of these non-essential Bam-associated proteins is not fully understood. Here, we analysed {Delta}bamB, {Delta}bamC, {Delta}bamE, {Delta}surA, {Delta}skp and {Delta}degP knockout strains by phenotypic screening, conservation analysis and high-throughput genetics. We identify hundreds of synthetic-lethal interactions and reveal that Bam complex activity is impacted by changes in outer membrane lipid composition and that enterobacterial common antigen is essential in the absence of the chaperone SurA. We also show genes responsible for synthesis of peptidoglycan are synthetically-lethal with Bam accessory lipoprotein encoding genes. Together, our data indicates potential mechanisms for coordination of OMP biogenesis with other cellular growth processes such as LPS and peptidoglycan biogenesis.

microbiology↗

bioRxiv2022

TraDIS-validate: a method for curating ordered gene-replacement libraries

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterised genes. To address this gap in functional annotation, many high-throughput screens have been devised with the goal of uncovering novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered gene-replacement library. We applied our method (TraDIS-Validate) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates. TraDIS-validate therefore had a success rate of 99.7% for identifying the correct kanamycin cassette position. This dataset provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of an antibiotic resistance cassette in any ordered library.

genetics↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2021

DpaA detaches Braun's lipoprotein from peptidoglycan

Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Brauns lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Brauns lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

microbiology↗