Biology subjects

Karambelkar, S.

Publications and source records attributed to Karambelkar, S..

4 recordsLinked to original sources

bioRxiv2026

A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

microbiology↗

bioRxiv2022

RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

The viruses that infect bacteria, bacteriophages (or phages), possess numerous genes of unknown function. Genetic tools are required to understand their biology and enhance their efficacy as antimicrobials. Pseudomonas aeruginosa jumbo phage {Phi}KZ and its relatives are a broad host range phage family that assemble a proteinaceous "phage nucleus" structure during infection. Due to the phage nucleus, DNA-targeting CRISPR-Cas is ineffective against this phage and thus there are currently no reverse genetic tools for this family. Here, we develop a DNA phage genome editing technology using the RNA-targeting CRISPR-Cas13a enzyme as a selection tool, an anti-CRISPR gene (acrVIA1) as a selectable marker, and homologous recombination. Precise insertion of foreign genes, gene deletions, and the addition of chromosomal fluorescent tags into the {Phi}KZ genome were achieved. Deletion of phuZ, which encodes a tubulin-like protein that centers the phage nucleus during infection, led to the mispositioning of the phage nucleus but surprisingly had no impact on phage replication, despite a proposed role in capsid trafficking. A chromosomal fluorescent tag placed on gp93, a proposed "inner body" protein in the phage head revealed a protein that is injected with the phage genome, localizes with the maturing phage nucleus, and is massively synthesized around the phage nucleus late in infection. Successful editing of two other phages that resist DNA-targeting CRISPR-Cas systems [OMKO1 ({Phi}KZ-like) and PaMx41] demonstrates the flexibility of this method. RNA-targeting Cas13a system holds great promise for becoming a universal genetic editing tool for intractable phages. This phage genetic engineering platform enables the systematic study of phage genes of unknown function and the precise modification of phages for use in a variety of applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/480438v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b1b4a6org.highwire.dtl.DTLVardef@cb0e08org.highwire.dtl.DTLVardef@1064217org.highwire.dtl.DTLVardef@1dcfbb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

The broad host range bacteriophage Mu employs a novel methylcarbamoyl modification to protect its DNA from diverse host restriction systems. Biosynthesis of the unusual modification is a longstanding mystery. Moreover, isolation of Mom, the phage protein involved in the modification has remained elusive to date. Here, we characterized the co-factor and metal binding properties of Mom and provide a molecular mechanism to explain methylcarbamoylation by Mom. Our computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom, predicting acetyl CoA as its co-factor. We demonstrate that Mom binds to acetyl CoA and identify the active site. Puzzlingly, none of the > 309,000 GNAT members identified so far catalyze Mom-like modification of their respective substrates. Besides, conventional acid-base catalysis deployed by typical acetyltransferases cannot support methylcarbamoylation of adenine seen in Mu phage. In contrast, free radical-chemistry, catalyzed by Fe-S cluster or transition metal ions can explain the seemingly challenging reaction between acetyl CoA and DNA. We discovered that Mom is an iron-binding protein, with the Fe2+/3+ ion colocalized with acetyl CoA in the active site of Mom. Mutants defective for binding Fe2+/3+ or acetyl CoA demonstrated compromised activity, indicating their importance in the DNA modification reaction. Iron-binding in the GNAT active site is unprecedented and represents a small step in the evolution of Mom from the ancestral acetyltransferase fold. Yet, the tiny step allows a giant chemical leap from usual acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture. SummaryStudying the arms race between bacteria and their viruses (bacteriophages or phages) is key to understanding microbial life and its complexity. An unprecedented DNA modification shields phage Mu from bacterial restriction endonucleases that destroy incoming phage DNA. Nothing is known of how the modification is brought about, except that a phage protein Mom is involved. Here, we discover acetyl CoA and iron as key requirements for the modification. We explain how by evolving the ability to bind iron - a transition metal capable of generating highly reactive free radicals, a well-studied scaffold like the acetyltransferase fold can gain novel catalytic prowess in Mom. These findings have broad implications for gene editing technologies and therapeutic application of phages.

biochemistry↗

bioRxiv2019

Listeria phages induce Cas9 degradation to protect lysogenic genomes

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse \"anti-CRISPR\" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional \"partner\" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an \"anti-anti-CRISPR.\" In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

microbiology↗