Munder, F. | Search bioRxiv

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2024

De novo design of potent CRISPR-Cas13 inhibitors

CRISPR-Cas systems are transformative tools for gene editing which can be tuned or controlled by anti-CRISPRs (Acrs) - phage derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that are capable of inhibiting biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful, rapid, and generalisable approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Using CRISPR-Cas13 as a representative example, we demonstrate that AI-designed anti-CRISPRs (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13 proteins. We present a comprehensive workflow for design validation and demonstrate AIcrs functionality in controlling CRISPR-Cas13 activity in bacteria. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture, and microbiology.

biochemistry↗

bioRxiv2024

High-affinity PQQ import is widespread in Gram-negative bacteria

PQQ is a soluble redox cofactor used by diverse bacteria to oxidise fuel compounds as a source of electrons for the respiratory chain. Many Gram-negative bacteria that encode PQQ-dependent enzymes do not possess the biosynthetic machinery for its production and instead obtain it from the environment. To achieve this the bacterium Escherichia coli uses the TonB-dependent transporter PqqU as a high-affinity PQQ importer, allowing it to use PQQ at an external concentration as low as 1 nM. Here, we show that PqqU achieves this by binding PQQ with a very high affinity. Using cryo-electron microscopy we determine the structure of the PqqU-PQQ complex at a resolution of 1.99 [A], revealing that the extracellular loops of PqqU undergo significant conformational changes upon PQQ binding, which captures the cofactor in an internal cavity. This cavity likely facilitates an airlock-style gating mechanism that prevents non-specific import through PqqU. Using structural modelling we show that the change in PqqU structure upon PQQ binding precludes the binding of bacteriophage, which targets it as a cell surface receptor. Guided by the PqqU-PQQ complex structure we use phenotypic analysis to identify the amino acids essential for PQQ import and leverage this information to map the presence of PqqU across Gram-negative bacteria. This reveals that PqqU is encoded by Gram-negative bacteria from at least 22 phyla from diverse habitats, including those found in aquatic, soil, host-associated, and extreme environments. This indicates that PQQ is a ubiquitous nutrient in many environments, and an important cofactor for bacteria that adopt diverse lifestyles and metabolic strategies. Significance StatementMany enzymes form complexes with molecules called cofactors to perform their function. PQQ is a cofactor used by bacterial enzymes that provide energy by breaking down food molecules. While some bacteria make their own PQQ, other bacteria use the transport protein PqqU to bind PQQ from the environment and import it into their cells. We show that PqqU binds PQQ very tightly, allowing bacteria to acquire it at very low concentrations. Using cryo-electron microscopy we image the PqqU-PQQ complex on an atomic level, revealing how PQQ is bound so tightly. Using this the information to analyse microbial genomes, we show that PQQ scavenging is employed by diverse bacteria, implying that PQQ is an important common good of diverse microbiomes.

biochemistry↗