Biology subjects

Choudhary, D. K.

Publications and source records attributed to Choudhary, D. K..

4 recordsLinked to original sources

bioRxiv2025

CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing inter-species mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favour of the targeting strain during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the MRE11-RAD50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.

genetics↗

bioRxiv2025

A TIR-SAVED effector mediates antiviral immunity via a conserved host signal

Cyclic oligonucleotide-based anti-phage signalling systems (CBASS) are widespread prokaryotic antiviral defense mechanisms that function through coordinated cyclase-effector interactions. Upon sensing viral infection, the cyclase produces a signal molecule that activates effector function and causes cell dormancy or death. However, the evolutionary origins and functional independence of CBASS components remain unclear. Type II CBASS systems commonly employ TIR-SAVED domain effector proteins that deplete cellular NAD+ during viral infection. Here, we demonstrate that a TIR-SAVED effector protein can operate as a standalone antiviral defense, causing significant growth inhibition and approximately 50% viral clearance during infection in the complete absence of its cognate cyclase. Remarkably, we show that the TIR-SAVED effector can sense cyclic di-AMP, a conserved second messenger produced by the host diadenylate cyclase DacZ, when the canonical CBASS signal is absent. This antiviral activity was associated with depletion of cellular NAD+ and required intact conserved functional residues within both the TIR and SAVED domains. These findings reveal a novel mechanism of antiviral signalling that expands the functional repertoire of CBASS. They also provide insights into the modular evolution of complex prokaryotic immune systems, suggesting that what are now CBASS effectors might have evolved as independent defense components before being integrated into multi-protein systems.

microbiology↗

bioRxiv2024

An archaeal CBASS system eliminates viruses without killing the host cells

Many cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) defend against viral infections by depleting cellular NAD+ levels, eventually leading to dormancy or death. This abortive infection strategy is beneficial in stopping fast lytic infections, as cells die before spreading the virus to neighbouring cells. However, in chronic viral infections, which often occur in archaea, abortive infection could be detrimental, as the cost of immunity may outweigh that of infection. Here we study an archaeal CBASS system (H-CBASS2) that was expressed in the model species Haloferax volcanii DS2 and Haloferax gibbonsii LR2-5. We show that the system provides protection to H. gibbonsii against a lytic tailed haloarchaeal virus HFTV1 by depleting NAD+, similar to what has been observed for lytic phages in bacteria. H-CBASS2 is also triggered, though with much slower activity, during infection with the chronic, non-lytic virus HFPV-1, and promotes virus clearance after several passages without killing host cells. Moreover, cells that clear the HFPV-1 infection become substantially more resistant to subsequent infections, due to mutations in envelope-associated proteins. Cell death by NAD+ depletion only occurs after a very long infection with HFPV-1 on solid medium. These findings suggest that the magnitude of H-CBASS2 response is somehow tuned to the infection type can benefit the host during non-lytic infections, potentially explaining why such systems are relatively common in archaea.

microbiology↗

bioRxiv2024

A new archaeal virus that suppresses the transcription of host immunity genes

In some extreme environments, archaeal cells have been shown to have chronic viral infections, and such infections are well-tolerated by the hosts and may potentially protect against more lethal infections by lytic viruses. We have discovered that a natural Haloferax strain (48N), which is closely related to the model organism Haloferax volcanii, is chronically-infected by a lemon-shaped virus, which we could purify from the medium. The chronic infection by this virus, which we named LSV-48N, is never cleared, despite the multiple defense systems of the host that include CRISPR-Cas, and two CBASS systems. Curing 48N of its virus by genetic engineering, led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of approximately 1 hour at 45{degrees}C compared to the typical 2.5 hours of H. volcanii or its infected isogen, and faster than any known haloarchaeon. The virus subverts host defenses by reducing their transcription and interfering with the CRISPR spacer acquisition machinery. Our results suggest that the slow growth of many halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.

microbiology↗