Biology subjects

Bhoobalan-Chitty, Y.

Publications and source records attributed to Bhoobalan-Chitty, Y..

5 recordsLinked to original sources

bioRxiv2025

A Pair of DNA Glucosyltransferases Elevate Counter-defense in Bacteriophage T4

Bacteriophages encode diverse pathways to modify their nucleobases. These modifications help phages to evade the host defense systems such as restriction-modification (RM), and type II and type V CRISPR-Cas systems. On the other hand, modifications can also serve as a target for other host defense systems, illustrating the complexity of the defense and counter-defense landscape. Bacteriophage T4 encodes two glucosyltransferases (GTs), -GT and {beta}-GT, that post-replicatively add a glucose moiety to the hydroxymethylated deoxycytosines (5-hmC) on phage DNA in the - and {beta}-conformation, respectively. Among all known phages, only six closely related phages encode both -GT and {beta}-GT. Here, through biochemical and genetic analysis, we show that {beta}-GT has higher catalytic activity, whereas -GT is more strongly expressed. During the T4 infection, these factors determine the contributions of both GTs, with -GT and {beta}-GT contributing respectively to glucosylation of 66% and 33% of all 5-hmC. Encoding a single GTs is sufficient for T4 to overcome the E. coli type I and type IV RM systems, unless the glucosylation capacity decreases below the 80% threshold. However, when encountering a host encoding DNA glycosylase Brig1 in addition to type I and type IV RM systems, a second GT is necessary to enable Brig1 escapers to resist RM systems. These results demonstrate that encoding multiple GTs with redundant functionalities provides an evolutionary advantage when simultaneously confronted with multiple antiphage defense systems.

microbiology↗

bioRxiv2024

Genetic manipulation of Bacteriophage T4 utilizing the CRISPR-Cas13b system

CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to the hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, -gt and {beta}-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. The T4 genome editing protocol was optimized to obtain recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this study presents a detailed description of the rapid and easy production of T4 phage specific mutants. IMPORTANCEThe use of phages for therapeutic purposes requires a complete understanding of their life cycle. For this purpose, its very useful to have a wide range of phage genome engineering tools at our disposal, each adapted to a particular phage or situation. Although T4 phage has been studied extensively over the past seven decades, a complete understanding of its lytic cycle is still lacking. Cas9- and Cpf1-dependent genome editing techniques for T4 have proven to be inconsistent due to the glucosyl-hydroxymethyl modification of the cytosine residues in its genome. The RNA targeting of the Cas13 system presents an ideal alternative, as demonstrated here, to overcome interference from DNA hypermodification. Apart from demonstrating a new genome editing technique in T4, we have generated a range of T4 variants demonstrating the efficacy of our technique in obtaining meaningful and difficult to construct phage mutants.

synthetic biology↗

bioRxiv2024

Multilayered interaction between CRISPR-Cas subtype I-A and adjacently encoded Acrs of virus SIRV2

Among the >100 anti-CRISPRs (Acrs) identified to date, the vast majority inhibit CRISPR-Cas immunity on its own. Here we report a multilayered interaction between CRISPR-Cas subtype I-A immunity and two Acrs encoded adjacently in the genome of Saccharolobus virus SIRV2, gp47 (AcrIA3) and gp48 (previously known as AcrIIIB1, hence termed AcrIIIB1/AcrIA4). The host subtype I-A CRISPR-Cas interference module was shown previously to be up-regulated upon SIRV2 infection, through the release of transcriptional repressor Csa3b from the promoter. We demonstrate that AcrIIIB1/AcrIA4 on its own increases viral infectivity 4-5 orders of magnitude in the presence of the host subtype I-A CRISPR-Cas immunity. This Acr is able to completely inhibit the subtype I-A CRISPR-Cas immunity when the transcriptional activation of the latter is artificially removed, suggesting that Acrs might be one of the driving forces for the evolution of CRISPR-Cas up-regulation. Interestingly, AcrIA3 cooperates with AcrIIIB1/AcrIA4 by inhibiting transcriptional activation of the host subtype I-A CRISPR-Cas interference module through interaction with the promoter of the latter. Taken together, our data shed light on how virus-host arms race shaped the evolution of CRISPR-Cas and Acrs.

microbiology↗

bioRxiv2023

To be or not to be an anti-CRISPR: AcrIII-1 and the importance of working with native biological systems

Viral members of the protein family DUF1874 have been reported to act as anti-CRISPR (acr) proteins that degrade cyclic tetra-adenylate (cA4), a nucleotide second messenger produced after the activation of several type III CRISPR-Cas systems in bacteria and archaea. Specifically, protein SIRV1 gp29 inhibits type III-A and type III-B CRISPR systems in plasmid-born assays in heterologous systems. In this work, we investigate the function of SIRV1 gp29 and its close homolog SIRV2 gp37 in a native biological context, i.e. in cultures infected by SIRV2. SIRV2 was selected instead of SIRV1 because the latter is not available any more from any laboratories. We demonstrate that gp37 has no anti-CRISPR activity during infection of Saccharolobus islandicus LAL14/1 with SIRV2, although it is able to protect SIRV2 from type III targeting when expressed from a plasmid. The inability of gp37 to act as an acr in the native, biological system is due to the protein expression timing: gp37 is a middle/late gene, thus unable to inhibit CRISPR-Cas targeting at the onset of infection. On the other hand, we find that while gp37 is a non-essential gene, it confers a mild replicative advantage to the virus. This advantage is mediated, in hosts with active CRISPR-Cas targeting, by the interaction between gp37 and host protein SiL_1451, which results in the inhibition of the lysine methyltransferase activity of SiL_1451, responsible for extensive methylation of surface lysines of two-thirds of the cellular proteins. Heterologous gene expression from a plasmid-borne non-native promoter has allowed the discovery and characterization of dozens of prokaryotic defense systems in recent years. Although this experimental strategy has several advantages, our study highlights the importance of validating relevant results under native conditions, and the limitations of extrapolating results obtained using heterologous systems.

microbiology↗

bioRxiv2021

Replication protein Rep provides selective advantage to viruses in the presence of CRISPR-Cas immunity

Prokaryotic viruses express anti-CRISPR (Acr) proteins to inhibit the host adaptive immune system, CRISPR-Cas. While the virus infection biology was shown to be strongly dependent on the relative strengths of the host CRISPR-Cas and viral Acrs, little is known about the role of the core processes of viral life cycle (replication, packaging etc) in defence/anti-defence arms race. Here, we demonstrate the selective advantage provided by a replication initiator, Rep, in the context of CRISPR-Acr interactions. First, we developed a two-host based CRISPR-Cas genome editing tool for the deletion of highly conserved and thus potentially important viral genes. Using this strategy, we deleted a highly conserved Rep-coding gene, gp16, from the genome of Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The knockout mutant ({Delta}gp16) produced around 4 fold less virus in a CRISPR-null host, suggesting that Rep is the major contributor to replication initiation in Rudiviridae. Indeed, DNA sequencing revealed Rep-dependent replication initiation from the viral genome termini, in addition to Rep-independent replication initiation from non-terminal sites. Intriguingly, the lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the {Delta}gp16 mutant much more quickly in CRISPR-containing host than in CRISPR-null host, demonstrating a selective advantage provided by Rep in the presence of host CRISPR-Cas immunity. Despite the non-essentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales. ImportanceCRISPR-Cas and anti-CRISPR proteins are accessary to prokaryotes and their viruses respectively. To date, research has been focused on their diversity, molecular mechanisms and application in genome editing. How CRISPR-Acr arms race influence the evolution of viral core genes involved in the basic virus life cycle remained a gap of knowledge so far. This study provides the first evidence that CRISPR-Acr arms race poses a selection pressure on the efficiency of viral genome replication, forcing viruses to evolve highly productive replication machineries..

microbiology↗