Biology subjects

Banzhaf, M.

Publications and source records attributed to Banzhaf, M..

At least 19 recordsLinked to original sources

bioRxiv2026

CAZyme Annotation of Five Parasitic Trichomonads Reveals a Conserved Repertoire of Host Glycan Targeting Proteins

Deciphering the functional potential of the unusually large and repetitive genomes of trichomonads has remained challenging since the first Trichomonas vaginalis genome assembly in 2007. These genomes are characterised by extensive gene duplication, widespread lateral gene transfer from prokaryotes, and numerous hypothetical proteins. Here, we systematically re-annotated five parasitic trichomonad genomes using the CAZy annotation pipeline, identifying 1,406 CAZyme-encoding genes (which encompass all four core CAZyme classes and carbohydrate-binding modules (CBMs)) - including 516 genes previously annotated as encoding hypothetical proteins - and generating predictions of CAZy genes originating from potential lateral gene transfer, encoding candidate secreted proteins, and their candidate metabolic pathway involvement. Comparative analyses revealed a conserved, highly duplicated repertoire of candidate mucin O-glycan-targeting CAZymes across all five species, alongside broad conservation of a candidate antibacterial CAZyme toolkit. Together, these findings substantially enrich functional annotation of these genomes and highlight in particular CAZymes and CBMs as promising targets for the future characterisation of the molecular and cellular basis of parasite-host and parasite-microbiota interactions underlying mucosal disease and dysbiosis associated with trichomonads.

evolutionary biology↗

bioRxiv2026

Restoring Antibiotic Sensitivity via Metabolic Pathway Manipulation in Pseudomonas aeruginosa

Stopping antimicrobial multidrug resistance (AMR) is essential to maintain the efficacy of current treatments and sustain modern medicine, but current control relies on a coordinated One Health approach involving stewardship, infection prevention and other control measures, without direct actionable mechanisms against resistance itself. Here, integrating proteomics and metabolomics with high-throughput Caenorhabditis elegans infection models, we show that unique synergy of biotin biosynthesis inhibition with metformin uncouples virulence from bacterial viability shutting down major Pseudomonas aeruginosa (PA) virulence mechanisms that impair host survival. Under directed evolution induced by central carbon metabolic stress, laboratory and clinical PA strain isolates acquire mutations in the MexAB-OprM multidrug efflux pump, leading to reversal of the AMR phenotype and restored susceptibility to multiple legacy and last-resort antibiotics such as meropenem. Our findings reveal a previously unexploited vulnerability in PA, its co-dependence on biotin biosynthesis and energy metabolism for virulence and resistance. This work establishes a conceptual framework in which metabolic co-targeting can abolish pathogenesis and drive reversal of antimicrobial resistance. It lays the foundation for first-in-class, non-antibiotic PA-selective anti-virulence and resistance-reversing therapies, key to extending the lifesaving duration of existing antibiotics.

microbiology↗

bioRxiv2026

Enriching the Human Stool Microeukaryotes for Shotgun Sequencing

The human gut microbiome harbours a diverse community of microeukaryotes, predominantly fungi, which may potentially play important roles in gut ecology and homeostasis. Despite their potential, the study of gut microeukaryotes has been hampered by the limited sensitivity of standard sequencing approaches, which struggle to capture DNA from low-abundance microorganisms against the overwhelming background of bacterial biomass. To address this, we developed a method to selectively enrich for microeukaryotic cells in human faecal samples by depleting bacterial cells prior to metagenomic sequencing. Through systematic comparison and optimisation at each processing step, we established a robust standard operating procedure (SOP) for microeukaryotic cell enrichment. By benchmarking this SOP across eight human faecal samples with three technical replicates each, we showed that it consistently increased microeukaryote representation in metagenomic libraries, greater microeukaryotic taxonomic diversity, and a reduced proportion of unclassified taxa. Together, these improvements enabled substantially deeper characterisation of the microeukaryotic fraction of the human gut microbiome.

microbiology↗

bioRxiv2026

A Type VII-secreted toxin enables inter-mycobacterial competition

Summary ParagraphMost bacteria live in complex environments where resources are scarce and competition is fierce. These organisms have evolved mechanisms to compete with other bacteria, often through the specialised secretion of proteinaceous toxins. Mycobacteria have not previously been reported to engage in this form of competition. The thick and unusual cell wall of mycobacteria, comprised of peptidoglycan, arabinogalactan and mycolic acids, is generally thought to be highly protective to these bacteria. Many enzymes have evolved to maintain this structure, including the GH183 family, which cleaves arabinogalactan. Here, we establish for the first time that some mycobacteria have weaponised a subset of these endo-D-arabinanases to enable inter-bacterial competition. We show that mycobacteria secrete an endo-D-arabinanase effector via the type VII secretion system that specifically targets the arabinogalactan layer of the Mycobacteriales cell envelope. Using structural biology and biochemistry, we identify the molecular basis for this activity and reveal a new protein family that protects the bacterium from the activity of this toxin. Finally, our data uncover widespread T7-secreted toxins in the Mycobacteriales, pointing to extensive inter-mycobacterial competition.

microbiology↗

bioRxiv2026

Chemical genomics screening reveals novel functions for unannotated genes in Vibrio cholerae C6706

Vibrio cholerae, the causative agent of cholera, remains a major global health burden despite ongoing initiatives for vaccination and improved sanitation. Much of the V. cholerae life cycle occurs in aquatic environments, requiring gene functions that enable survival under diverse stresses associated with this niche and during the transition to the human host. Although numerous V. cholerae genome sequences are available, functional annotation across its pan-genome remains incomplete, and many annotated genes lack experimental validation. In this study, we employed a chemical genomics approach to profile fitness phenotypes across 104 diverse stress conditions using a library of 3,026 single-gene mutants of V. cholerae C6706, a widely used research strain. In total, we identified significant growth phenotypes for 1,518 mutants, of which 88 correspond to unannotated (hypothetical or putative) genes. Together, these data provide a comprehensive resource for exploring gene function in V. cholerae, supported by detailed quality metrics and open-access tools to facilitate community-driven discovery. AUTHOR SUMMARYAlthough many bacterial pathogens have been extensively sequenced, we still know relatively little about what many of their genes actually do. In many cases, gene functions are solely predicted by computer algorithms but have not been confirmed in the laboratory, and we can assume that some of those predictions may be incorrect. Chemical genomics -- a method that tests thousands of single-gene mutants under different stresses -- can help reveal what these genes are responsible for. Using this approach, we screened a library of Vibrio cholerae C6706 mutants under 104 different conditions. We found that more than half of the mutants showed measurable effects on growth, including many genes that were previously uncharacterized. Here, we describe our experimental pipeline, present key quality checks, and share how other researchers can use this dataset as a resource to explore V. cholerae mutant phenotypes.

systems biology↗

bioRxiv2026

Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium Zymomonas mobilis

The alpha-proteobacterium Zymomonas mobilis exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular, Z. mobilis has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualise its cellular localisation and estimate chromosome copy number in individual cells. Imaging showed that Z. mobilis exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, demonstrating that a single Z. mobilis cell contains at >5 copies of the chromosome at the oriC regions. After verifying its polyploidy, we sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in Z. mobilis. We found that, despite Z. mobilis secreting a native extra-cellular sucrase SacB, heterologous periplasmically-localised Bacillus subtilis SacB rendered Z. mobilis cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the Z. mobilis genome. Together, this work provides important insights and tools for advancing Z. mobilis genetics and its biotechnological applications. ImportanceZymomonas mobilis is a promising industrial bacterium with capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual Z. mobilis cells carry multiple copies of chromosome as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that single Z. mobilis cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in Z. mobilis, enabling efficient and complete mutation of all chromosome copies.

microbiology↗

bioRxiv2025

ChemGenXplore: An Interactive Tool for Exploring and Analysing Chemical Genomic Data

MotivationChemical genomics is a useful high-throughput approach to systematically link phenotypes to genotypes. However, the vast datasets generated remain challenging to explore due to the lack of integrated, interactive tools for visualisation and analysis. Existing workflows often require multiple independent software tools, limiting data accessibility and collaboration. Therefore, we created a user-friendly platform that enable efficient exploration and sharing of chemical genomics data. ResultsWe developed ChemGenXplore, a web-based Shiny application designed to streamline the visualisation and analysis of chemical genomic screens. It offers two primary functionalities: one for exploring pre-implemented datasets and another for analysing user-uploaded datasets. ChemGenXplore enables users to visualise phenotypic profiles, assess gene-gene and condition-condition correlations, perform GO and KEGG enrichment analysis, and generate customisable, interactive heatmaps. By consolidating these features into an interactive and accessible tool, ChemGenXplore facilitates data sharing, enhances reproducibility, and promotes collaboration within the research community. Availability and ImplementationChemGenXplore is freely accessible as a web application at https://chemgenxplore.kaust.edu.sa/ and is also available for local deployment via GitHub at https://github.com/Hudaahmadd/ChemGenXplore.

bioinformatics↗

bioRxiv2025

An evolutionarily conserved laterally acquired toolkit enables microbiota targeting by Trichomonas

Trichomonas species are a diverse group of obligate extracellular symbionts associated with or attributed to various inflammatory diseases. They colonise mucosal surfaces across a wide range of hosts, all of which harbour a resident microbiota. Their evolutionary history likely involved multiple host transfers, including zoonotic events from columbiform birds to mammals. Using comparative transcriptomics, this study examines Trichomonas gallinae co-cultured with Escherichia coli, identifying a molecular toolkit that Trichomonas species may use to interact with bacterial members of the microbiota. Integrating transcriptomic data with comparative genomics and phylogenetics revealed a conserved repertoire of protein-coding genes likely acquired through multiple lateral gene transfers (LGT) in a columbiform-infecting ancestor. These LGT-derived genes encode muramidases, glucosaminidases, and antimicrobial peptides--enzymes and effectors capable of targeting bacterial cell walls, potentially affecting the bacterial microbiota composition across both avian and mammalian hosts. This molecular toolkit suggests that Trichomonas species can actively compete with and exploit their surrounding microbiota for nutrients, potentially contributing to the dysbiosis associated with Trichomonas infections. Their ability to target bacterial populations at mucosal surfaces provides insight into how Trichomonas species may have adapted to diverse hosts and how they could influence inflammatory mucosal diseases in birds and mammals.

molecular biology↗

bioRxiv2024

Wild-type and single-O-antigen repeat outer-membrane vesicles induce equivalent protection against homologous and heterologous Salmonella challenge

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S. Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), [≤]1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia.

immunology↗

bioRxiv2023

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves -1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

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↗

bioRxiv2023

ChemGAPP; A Package for Chemical Genomics Analysis and Phenotypic Profiling.

High-throughput chemical genomic screens produce informative datasets, providing valuable insights into unknown gene function on a genome-wide level. However, there is currently no comprehensive analytic package publicly available. We developed and benchmarked ChemGAPP to bridge this gap. ChemGAPP allows integration of various steps in a streamlined and user-friendly format, including rigorous quality control measures to curate screening data. ChemGAPP provides three sub-packages for different chemical-genomic screens: ChemGAPP Big for handling large-scale high-throughput screens; ChemGAPP Small, designed for small-scale screen analysis and ChemGAPP GI for genetic interaction screen analysis. ChemGAPP is available at https://github.com/HannahMDoherty/ChemGAPP.

bioinformatics↗

bioRxiv2022

Plasmid Permissiveness of Wastewater Microbiomes can be Predicted from 16S rDNA sequences by Machine Learning

Wastewater Treatment Plants (WWTPs) contain a diverse microbial community with high cell density. They constantly receive antimicrobial residues and resistant strains and, therefore, may offer conditions for the Horizontal Gene Transfer (HGT) of antimicrobial resistance determinants, transmitting clinically important genes between, e.g., enteric and environmental bacteria and vice versa. Despite the clinical importance, tools for predicting HGT are still under-developed. In this study, we examined to which extent microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e., the ability of cells to receive a plasmid through conjugation, for microbial communities in the water cycle, using data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, translating to the range of hosts a plasmid is able to transfer to, for three broad host-range resistance plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.45 for pB10 (95% CI: 0.42-0.52), 0.42 for pKJK5 (0.95% CI: 0.38-0.45) and 0.52 for RP4 (0.95% CI:0.45-0.55) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite these being broad host-range plasmids. Our results provide a framework that contributes to assessing the risk of AMR pollution in wastewater systems. The predictive tool is available as a an application under https://github.com/DaneshMoradigaravand/PlasmidPerm.

bioinformatics↗

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↗

bioRxiv2020

Global query on bacterial sirtuin CobB interactants reveals crosstalk with PRPP synthase Prs

In bacteria, post-translational acetylation of lysine residues affects the activities of numerous proteins, thus constituting an important regulatory mechanism that influences bacterial growth and adaptation to various environments. Conversely, the modification level of some lysine residues is controlled by deacetylases that remove the acetyl group. Most bacterial deacetylases are homologous to eukaryotic sirtuins, which utilize NAD+ as a cofactor. The Escherichia coli genome encodes a single sirtuin - CobB. However, both the regulation of CobB activity and the role that reversible acetylation plays in the control of protein functions and cellular pathways are not fully understood. In this study, we demonstrate that CobB forms a stable complex with PRPP synthase Prs. PRPP synthase is responsible for providing precursor metabolite for the synthesis of certain amino acids as well as nucleotides, including the cofactor NAD+. This association stimulates the deacetylation rate by CobB and protects it from inhibition by its reaction byproduct nicotinamide. We present evidence indicating that acetylation of Prs is physiologically significant and impacts E. coli metabolism and global protein acetylation level, although it does not affect CobB binding in vitro. In turn, Prs can be deacetylated by CobB, while formation of CobB-Prs complex formation safeguards PRPP synthase activity under non-optimal conditions. We propose that formation of the complex regulates deacetylation of other protein substrates by CobB.

molecular biology↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗

bioRxiv2020

Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 [A], in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. ImportanceM48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the LPS biogenesis machinery. Here we present the structure of BepA and the results of a structure guided mutagenesis strategy, which reveal the key residues required for activity.

microbiology↗

bioRxiv2020

Midcell localization of PBP4 of Escherichia coli is essential for the timing of divisome assembly

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. The hydrolytic enzymes outnumber the enzymes that insert new PG by far and very little is known about their specific function. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that domain 3 is needed for the interaction with NlpI, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that PBP4 functions together with the amidases AmiA and B to create denuded glycan strands to attract the initiator of septal PG synthesis, FtsN. Consistent with this model, we found that the divisome assembly at midcell was significantly affected in cells lacking PBP4. IMPORTANCEPeptidoglycan biosynthesis is a major target for antibacterials. The covalently closed peptidoglycan mesh, called sacculus, protects the bacterium from lysis due to its turgor. Sacculus growth is facilitated by the balanced activities of synthases and hydrolases, and disturbing this balance leads to cell lysis and bacterial death. Because of the large number and possible redundant functions of peptidoglycan hydrolases, it has been difficult to decipher their individual functions. In this paper we show that the DD-endopeptidase PBP4 localizes at midcell during septal peptidoglycan synthesis in Escherichia coli and is important for the timing of the assembly of the division machinery. This shows that inhibition of certain hydrolases could weaken the cells and might enhance antibiotic action.

microbiology↗