Biology subjects

Maxwell, K. L.

Publications and source records attributed to Maxwell, K. L..

10 recordsLinked to original sources

bioRxiv2026

A novel virus lineage is abundant in metaviromes from Dehalococcoides-containing mixed cultures

Dehalococcoides mccartyi are obligately anaerobic organohalide-respiring bacteria that play important roles in the detoxification of chlorinated pollutants in groundwater and sediments. Despite having small genomes, they host a diverse set of mobile elements. Here we characterize a family of mobile elements, termed integrative and mobilizable element 1 or IME1, comprising 20 from Dehalococcoides and one from Dehalogenimonas alkenigignens. IME1s are 20,930 - 28,058 bp and are found both integrated in the genomes and as circular episomes. Bioinformatic characterization of IME1 encoded proteins revealed a highly conserved structure with 14 hierarchical orthologous groups (HOGs) found in all 21 IME1s. IME1s lack recognizable hallmark proteins of tailed bacterial viruses (or tailed phages) but encode proteins with similarities to those of filamentous bacterial viruses. In particular, one conserved HOG shows sequence similarity to the pI-like ATPase, the only conserved marker protein identified across filamentous bacterial viruses. Additionally, IME1s encode several small proteins with predicted transmembrane domains and signal peptides, another feature used to identify filamentous bacterial viruses. Both features are also found in budding archaeal viruses of various morphotypes. IME1s dominated metaviromes obtained from the Dehalococcoides-containing KB-1 mixed culture and electron micrographs of the corresponding viral fractions revealed abundant filamentous virus-like particles. We therefore propose that the IME1s represent a novel lineage of double stranded budding, likely filamentous, viruses. Database searches suggest IME1s are found in Dehalococcodia and other Chlorofexota but are so far restricted to this phylum.

microbiology↗

bioRxiv2026

A broad-spectrum phage-encoded mechanism to disarm bacterial type IV filaments

Phages can modify host cell physiology to thwart competitors. The Pseudomonas aeruginosa-specific phage DMS3 encodes Aqs1, a protein inhibitor of type IV pilus (T4P) function to prevent host cell recognition by other phages that leverage these filaments for infection. Aqs1 disrupts T4P by binding to the hexameric ATPase PilB, required to power pilus filament extension, though several mechanistic details remain unclear. We show that Aqs1 has broad-spectrum activity and can disrupt T4P function in a variety of Gram negative bacteria. This protein inhibits PilB by binding to a solvent-exposed hydrophobic patch on the N2-domain, distal to the active site. Binding destabilizes the hexamer, preventing PilB accumulation at T4P machines. Aqs1 likely disrupts PilB oligomerization by displacing a flexible linker segment between the PilB N1- and N2-domains required for inter-subunit contact. Together, the Aqs1 mode of action provides a design template for broad-spectrum inhibitors of diverse bacterial virulence factors. SignificanceThe phage-encoded protein Aqs1 disables type IV pilus (T4P) production in Pseudomonas aeruginosa by targeting the hexameric ATPase responsible for assembling pilus fibers. We show that despite originating from a P. aeruginosa-specific phage, Aqs1 can also disable T4 ATPase-dependent phenotypes across other pathogenic bacteria and homologous systems. Mechanistically, Aqs1 binds to a conserved patch on the PilB N2-domain away from the active site. Binding here breaks apart the PilB oligomer, preventing it from acting on T4P machines. Aqs1 binding at the N2-domain patch likely displaces a flexible PilB linker segment that binds to this site to stabilize the hexamer. Our work highlights a novel and conserved PilB allosteric site which is exploited by the phage-encoded protein Aqs1 to disrupt diverse T4 systems in multiple bacteria.

microbiology↗

bioRxiv2025

Antiviral Defence is a Conserved Function of Diverse DNA Glycosylases

Bacteria are frequently attacked by viruses, known as phages, and rely on diverse defence systems like restriction endonucleases and CRISPR-Cas to survive. While phages can evade these defences by covalently modifying their DNA, these non-canonical nucleobases create a strong selective pressure for host proteins that can recognize and exploit them. Here, using a structure-guided discovery approach, we identify widespread families of DNA glycosylases that protect bacteria against phages that incorporate modified guanine bases into their DNA. Despite high sequence variation, these enzymes share a conserved glycosylase fold and occur across bacterial lineages. We also uncover a distinct glycosylase superfamily that defends against phages with thymidine modifications, showing that glycosylases have repeatedly evolved as antiviral defences. Together, these findings reveal DNA glycosylases as versatile effectors of bacterial immunity and underscore structure-guided discovery as a powerful strategy for uncovering hidden layers of antiviral defence.

microbiology↗

bioRxiv2024

Molecular basis of foreign DNA recognition by BREX anti-phage immunity system

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on epigenetic DNA methylation to discriminate between the host and invading DNA, but their mechanism of protection remains enigmatic. We demonstrate that in Type I BREX systems, both defense and methylation are based on site-specific DNA recognition by the BrxX (PglX) methyltransferase and require the S-adenosyl methionine cofactor. We present a 2.2-[A] cryoEM structure of Escherichia coli BrxX bound to target dsDNA, which reveals the molecular details of DNA recognition by BREX and paves the way for rational engineering of BREX specificity. We show that BrxX alone does not support methylation, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters an inactive dimeric form of BrxX. Together, these results allow us to propose a model of BREX-mediated DNA sensing and anti-phage defense.

molecular biology↗

bioRxiv2024

Prophages express a type IV pilus component to provide anti-phage defence

Phage genomes integrated within bacterial genomes, known as prophages, frequently encode proteins that provide defence against further phage infection. These proteins often function at the cell surface and prevent phages from attaching to their host receptor. Here, we describe the discovery of prophage-encoded proteins that resemble FimU, a component of the Pseudomonas aeruginosa type IV pilus. These phage FimU proteins are incorporated into the pilus without altering its function, yet they mediate robust protection against infection by phages that bind to the tip of the pilus, where FimU is likely located. The phage FimU proteins and the phage tail proteins that interact with FimU are highly diverse, suggesting that evolution in this system is driven by phage versus phage competition. To our knowledge, this is the first example of anti-phage defence mediated by replacement of a bacterial cell surface component with a phage-encoded protein.

microbiology↗

bioRxiv2024

Prophages block cell surface receptors to ensure survival of their viral progeny

In microbial communities, viruses compete for host cells to infect, and thus evolved diverse ways to inhibit their competitors. One mechanism is Superinfection exclusion (Sie), whereby a virus that has established an infection prevents a secondary infection. We identified a Pseudomonas prophage Sie protein that alters pilus dynamics through the pilus assembly chaperone, PilZ. This protein, known as Zip for PilZ interacting protein, does not abrogate pilus activity, but fine tunes it, providing strong phage resistance without a fitness cost. This tuning is modulated through quorum sensing, which coordinates Zip production in concert with bacterial cell density to ensure maximal protection when bacterial populations are at the highest risk of phage infection. Most notably, Zip activity prevents internalization and destruction of phage progeny. We refer to this as the "anti-Kronos effect" after the Greek god who devoured his own children and show that it is a conserved feature of diverse prophage-encoded Sie systems.

microbiology↗

bioRxiv2023

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages. Abbreviated summaryQuorum sensing regulates phage defense in Pseudomonas aeruginosa. The Pf phage protein PfsE inhibits PQS-mediated quorum sensing by binding to the host enzyme PqsA, while also protecting against type IV pili-dependent phage infection. This dual inhibition strategy promotes Pf replication and safeguards the host from competing phages.

microbiology↗

bioRxiv2021

Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

biochemistry↗

bioRxiv2021

F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

microbiology↗

bioRxiv2018

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2-4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

molecular biology↗