Biology subjects

Costa, T. R. D.

Publications and source records attributed to Costa, T. R. D..

4 recordsLinked to original sources

bioRxiv2026

Relaxase Asymmetry Drives Initiation of Bacterial Conjugation

Bacterial conjugation is the major route of horizontal gene transfer and a key driver of the dissemination of antibiotic resistance, exacerbating the global health crisis. Central to this process is TraI, a bifunctional relaxase-helicase encoded by the iconic F-plasmid family. TraI initiates single-stranded DNA (ssDNA) transfer by covalently attaching to plasmid DNA through its trans-esterase (TE) activity and subsequently unwinds the plasmid via its helicase activity. Although genetic and biochemical studies have postulated that two TraI molecules are required for efficient conjugative transfer, how these molecules coordinate their activities in space and time - and how strand nicking is coupled to helicase loading - has remained unknown. Here, we capture the elusive, functionally asymmetric TraI homodimer and report its cryo-EM structure, revealing that the interaction between the two TraI molecules is mediated predominantly by ssDNA. We further show that TE-driven duplex melting at the origin of transfer (oriT) by one TraI molecule enables helicase loading by a second TraI molecule on the transfer strand of the unnicked plasmid DNA. Although the TE domain is intrinsically competent for strand nicking, its activity is inhibited by the host factor IHF, and this inhibition is relieved upon helicase loading. Together, these findings define a regulatory loop that coordinates helicase loading with strand nicking, providing mechanistic insight into the earliest stages of conjugative DNA processing.

microbiology↗

bioRxiv2025

RfaH is Essential for Virulence and Adaptive Responses in Yersinia pseudotuberculosis Infection

We previously demonstrated that increased expression of the gene encoding transcriptional antiterminator RfaH during Yersinia pseudotuberculosis transcriptional reprogramming necessary for adapting to persistent infection. RfaH is known to regulate expression of the O-antigen biosynthesis operon in Y. pseudotuberculosis. In this study, we examined the role of RfaH in virulence, bacterial physiology under infection-relevant stress conditions, and determined the RfaH regulon in Y. pseudotuberculosis. We employed a mouse infection model and phenotypic assays to test RfaHs role in virulence and physiology, as well as RNA sequencing, including O-antigen biosynthesis-deficient strains. Our findings demonstrate that loss of rfaH significantly attenuates virulence, reducing the capacity of Y. pseudotuberculosis to establish persistent infection. RfaH expression is increased during the stationary growth phase and under various stress conditions, such as high osmolarity and temperature. Functional assays revealed that the{Delta} rfaH strain displayed defects in swimming and increased clumping, indicating altered surface properties affecting motility. Transcriptomic analysis showed that the absence of rfaH led to downregulation of genes involved in virulence besides O-antigen biosynthesis operon, suggesting RfaHs critical role in virulence and host adaptation. Notably, we identified a hypothetical non-coding RNA encoded within the 5-UTR of the O-antigen biosynthesis operon, which may regulate gene expression of the urease operon in Y. pseudotuberculosis. Collectively, our findings suggest that RfaH is essential for the virulence and adaptive capacity of Y. pseudotuberculosis to colonize the host. This study provides insights into regulatory mechanisms that facilitate bacterial survival in hostile environments and highlights the importance of RfaH and its regulatory targets in the pathogenesis of Y. pseudotuberculosis. Author SummaryFor bacterial pathogens to establish infection and persist in the host, they must adapt to harsh environments and fine-tune gene expression accordingly. The transcriptional antiterminator RfaH plays a pivotal role in regulating key genes essential for adaptation and virulence in Y. pseudotuberculosis. In this study, we explored the function of RfaH in bacterial physiology, stress responses, and infection dynamics. Using a mouse infection model, we found that loss of RfaH significantly reduced virulence and impaired the pathogens ability to establish persistent infection. Notably, RfaH expression increased under stress conditions, such as high osmolarity and temperature, underscoring its role in bacterial adaptation. On the other hand, the absence of RfaH led to motility defects and enhanced bacterial aggregation, suggesting alterations in surface properties. Transcriptomic analysis revealed that RfaH influences a broader set of genes beyond the O-antigen biosynthesis operon, including virulence factors critical for host adaptation. Additionally, we identified a potential non-coding RNA within the 5'-UTR of the O-antigen biosynthesis operon, which may regulate urease operon. Overall, our findings establish RfaH as a key regulator of Y. pseudotuberculosis virulence, shedding light on the molecular mechanisms that enable bacterial survival in challenging environments.

microbiology↗

bioRxiv2025

AI mirrors experimental science to uncover a novel mechanism of gene transfer crucial to bacterial evolution

AI models have been proposed for hypothesis generation, but testing their ability to drive high-impact research is challenging, since an AI-generated hypothesis can take decades to validate. Here, we challenge the ability of a recently developed LLM-based platform, AI co-scientist, to generate high-level hypotheses by posing a question that took years to resolve experimentally but remained unpublished: How could capsid-forming phage-inducible chromosomal islands (cf-PICIs) spread across bacterial species? Remarkably, AI co-scientists top-ranked hypothesis matched our experimentally confirmed mechanism: cf-PICIs hijack diverse phage tails to expand their host range. We critically assess its five highest-ranked hypotheses, showing that some opened new research avenues in our laboratories. We benchmark its performance against other LLMs and outline best practices for integrating AI into scientific discovery. Our findings suggest that AI can act not just as a tool but as a creative engine, accelerating discovery and reshaping how we generate and test scientific hypotheses.

microbiology↗

bioRxiv2025

Chimeric infective particles expand species boundaries in phage inducible chromosomal island mobilization

Some mobile genetic elements spread among unrelated bacterial species through unknown mechanisms. Recently, we discovered that identical capsid-forming phage-inducible chromosomal islands (cf-PICIs), a new family of phage satellites, are present across multiple species and genera, raising questions about their widespread dissemination. Here we have identified and characterized a new biological entity enabling this transfer. Unlike other satellites, cf-PICIs produce their own capsids and package their DNA, relying solely on phage tails for transfer. Remarkably, cf-PICIs release non-infective, tail-less capsids containing their DNA into the environment. These subcellular entities then interact with phage tails from various species, forming chimeric particles that inject DNA into different bacterial species depending on the tail present. Additionally, we elucidated the structure of the tail-less cf-PICIs and the mechanism behind their unique capsid formation. Our findings illuminate novel mechanisms used by satellites to spread in nature, contributing to bacterial evolution and the emergence of new pathogens.

microbiology↗