Biology subjects

McLean, T. C.

Publications and source records attributed to McLean, T. C..

8 recordsLinked to original sources

bioRxiv2026

TrbA binds and locks a sliding clamp KorB to repress transcription on multi-drug resistance plasmids

Precise regulation of gene expression is essential for all living systems. Genes encoded on mobile genetic elements such as conjugative plasmids are also highly regulated to ensure stable inheritance, successful horizontal transfer, and adaptation to diverse bacterial hosts. The multi-drug resistance plasmid RK2 encodes an intricate regulatory network centred on the ParB-CTPase family protein KorB, which functions in both plasmid segregation and global gene regulation. We recently showed that KorB acts as a CTP-dependent DNA-sliding clamp that can be converted into a transcriptional repressor through direct interaction with the DNA-binding protein KorA. This clamp sliding-and-locking mechanism by KorAB enables highly effective transcriptional repression. Here, we investigate whether a third transcriptional regulator on the RK2 plasmid, TrbA, operates through a similar mechanism. Using a combination of structural prediction, biochemistry and in vivo transcriptional assays, we provide evidence that TrbA also directly interacts with KorB and functions as a clamp-locking factor. This KorB-TrbA interaction is mediated by a conserved aromatic interface that enables strong cooperative transcriptional repression. Disruption of this interface abolishes synergy, indicating that direct protein-protein interaction underpins KorB-TrbA cooperativity. Finally, bioinformatic analysis of a large plasmid database reveal that the tripartite KorB-KorA-TrbA system is present on numerous plasmids. Together, our findings establish TrbA as a bona fide KorB co-repressor and demonstrate how a single sliding clamp protein can integrate multiple partners to evolve a complex transcriptional regulatory network. IMPORTANCEPrecise regulation of gene expression ensures gene products are produced at the right time and in the right amounts. Recent works uncovered a new mechanism of bacterial gene regulation based on a clamp sliding and locking in a multi-drug resistance plasmid, RK2. KorB functions as a CTP-dependent DNA-sliding clamp capable of traveling over a long genomic distance. Sliding KorB is captured and locked in place by a partner protein, KorA, forming a stable complex at target promoters to repress transcription. Here, we show that another RK2 regulator, TrbA, also use this clamp sliding-locking mechanism, and identify an aromatic interface enabling TrbA-KorB-mediated transcriptional repression. Our findings show how a single sliding clamp integrates multiple partners to build a complex transcriptional regulatory network.

microbiology↗

bioRxiv2026

Molecular basis for the role of Ripr in Plasmodium falciparum invasion of human erythrocytes

Plasmodium falciparum causes the majority of severe malaria, and merozoite invasion of erythrocytes is a vulnerable, antibody-accessible step of the blood-stage cycle. PfRipr is an essential component of the PCRCR invasion complex, yet the structural basis for antibody-mediated neutralisation remains unclear. Here, we map inhibitory and non-inhibitory epitopes across PfRipr and show that all potent inhibitors localise to the tail region (EGF6-8). Crystal structures reveal that inhibitory antibodies restrict the flexibility surrounding EGF7. Indeed, EGF7 buried surface area correlates strongly with inhibitory potency, identifying this domain as the principal invasion-inhibitory determinant. Pairwise antibody combinations revealed unexpected synergy, with non-inhibitory mAbs potentiating anti-Rh5 activity. Conditional deletion, sequence replacement or positional swapping of EGF6-8 abolished invasion, demonstrating that both sequence and spatial arrangement are indispensable. These data define EGF7 as a conserved, functionally essential vulnerability and provide a blueprint for rational EGF6-8 immunogen design capable of eliciting P. falciparum strain-transcending protection against blood-stage malaria.

biochemistry↗

bioRxiv2026

Repurposing a chromosome segregation ParB-CTPase fold into an ATPase toxin for contact-dependent growth inhibition in plant and animal pathogens

Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation.

microbiology↗

bioRxiv2025

Stable inheritance of the Streptomyces linear plasmid SCP1 by dual ParABS partition systems

Low-copy-number plasmids often rely on dedicated maintenance mechanisms, such as partitioning systems, to ensure stable inheritance across generations. These partition systems actively segregate sister plasmid copies during cell division and are classified by the NTPase types they encode. While the distribution and organization of partition system types are well characterized in Enterobacteriaceae plasmids, their functions and diversity across broader bacterial taxa remain poorly understood. Here, we analyze a large and diverse plasmid database to examine the distribution of partition system types and find that plasmids encoding multiple partition systems are more common than previously recognized. Notably, many plasmids encode multiple partition systems of the same type, an organization that has not been previously investigated. To further investigate, we employ the Streptomyces linear plasmid SCP1, which encodes two type I ATP- and CTP-dependent parABS partition systems, as a model. Sequence analysis shows that both SCP1-encoded ParBs harbor less conserved CTPase domains than their chromosomal counterparts, suggesting they might diverge from canonical ParB functions. However, using chromatin immunoprecipitation with deep sequencing, biochemical assays, and targeted mutagenesis, we demonstrate that both proteins are bona fide ParB CTPase proteins: they recognize distinct parS sites on SCP1, bind and hydrolyze CTP, and slide to accumulate on DNA. Despite both systems being functional, only parABS1, but not parABS2, is crucial for SCP1 maintenance under standard laboratory conditions. Altogether, these findings provide the first functional characterization of dual ParB-CTPase partition systems coexisting on a single plasmid, advancing our understanding of plasmid maintenance in Streptomyces, and reveal new aspects of the diversity and distribution of plasmid partition systems in bacteria.

microbiology↗

bioRxiv2025

Extracellular redox sensors control the protein secretion stress response in Streptomyces

Streptomyces secondary metabolites account for over half of all clinically used antibiotics, as well as numerous antifungal agents, anticancer compounds, and immunosuppressants. Two-component systems, which are widespread in bacteria, are key regulators of antibiotic production in Streptomyces species, yet their activating signals remain poorly understood. CutRS was the first two-component system identified in the genus Streptomyces and deletion of cutRS in Streptomyces coelicolor was shown to enhance antibiotic production, although its CutR regulon does not include any biosynthetic genes. Here, we used Streptomyces venezuelae to further investigate CutRS function. We show that deletion of cutRS leads to an increase in growth rate and a reversal of the typical glucose-mediated carbon catabolite repression typically observed in Streptomyces species. We also demonstrate that CutR DNA binding is glucose-dependent, but CutR does not directly regulate genes involved in growth, antibiotic biosynthesis, or glucose metabolism. The only CutR targets conserved in both S. coelicolor and S. venezuelae are the foldase genes htrA3 and htrB, which are involved in the protein secretion stress response. Consistent with this, we show that CutS homologues all contain two conserved cysteine residues in their extracellular sensor domains and that changing these residues to serine constitutively activates S. venezuelae CutRS. We propose that failure of a disulfide bond to form between these cysteine residues indicates secretion stress and leads to activation of the CutRS system and the secretion stress response. IMPORTANCEStreptomyces bacteria are the primary source of clinically useful antibiotics. While many two-component systems have been linked to antibiotic biosynthesis in Streptomyces species, few have been well characterized. Here, we characterize a secretion stress sensing two-component system called CutRS and propose a model for how the sensor kinase detects extracellular protein misfolding via two highly conserved cysteine residues. Importantly, we also show that deletion of cutRS triggers antibiotic overproduction in the presence of glucose. Since glucose normally represses antibiotic biosynthesis in Streptomyces species through carbon catabolite repression, this finding reveals a simple genetic route to bypass this barrier. This has significant implications for antibiotic discovery pipelines and industrial production, where glucose-rich media are preferred for cost and scalability. Our results position CutRS as a key target for future strain improvement strategies.

microbiology↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2024

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136{Delta}upf31 as well as the closely related archetypal IncP-1{beta} plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

microbiology↗

bioRxiv2024

LazyAF, a pipeline for accessible medium-scale in silico prediction of protein-protein interactions

Artificial intelligence has revolutionized the field of protein structure prediction. However, with more powerful and complex software being developed, it is accessibility and ease of use rather than capability that is quickly becoming a limiting factor to end users. Here, I present a Google Colaboratory-based pipeline, named LazyAF, which integrates the existing ColabFold BATCH to streamline the process of medium-scale protein-protein interaction prediction. I apply LazyAF to predict the interactome of the 76 proteins encoded on a broad-host-range multi-drug resistance plasmid RK2, demonstrating the ease and accessibility the pipeline provides. AvailabilityLazyAF is freely available at https://github.com/ThomasCMcLean/LazyAF

bioinformatics↗