Biology subjects

Rojas, A. A. R.

Publications and source records attributed to Rojas, A. A. R..

3 recordsLinked to original sources

bioRxiv2024

A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and an oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.

synthetic biology↗

bioRxiv2024

T4 phage RNA is NAD-capped and alters the NAD-capepitranscriptome of Escherichia coli during infection through a phage-encoded decapping enzyme

Nicotinamide adenine dinucleotide (NAD+) serves as a cap-like structure on cellular RNAs (NAD-RNAs) across all domains of life, including Escherichia coli. Beyond its role in metabolism, NAD+ also functions as a regulatory molecule in phage defense mechanisms. However, NAD-RNAs have not yet been identified during bacteriophage infections, and the mechanisms governing their synthesis and degradation in this context remain unknown. To address this gap, we used T4 phage infection of E. coli as a defined and well-characterized model system to study NAD-RNAs at the virus-host interface. Here, we report the first identification and characterization of NAD-RNAs during phage infection. Using time-resolved NAD captureSeq, we identified NAD-capped host and T4 phage transcripts and observed that the set of enriched NAD-capped RNAs varies across different infection phases. Importantly, NAD captureSeq identifies NAD-capped transcripts based on enrichment and does not directly report the fraction of molecules that are NAD-capped. Consequently, temporal changes in enrichment may reflect altered NAD-capping, altered transcript abundance, or a combination of both. We provide evidence that NAD-RNAs are generated by the host RNA polymerase by initiating transcription with NAD+ at canonical transcription start sites. By quantifying intracellular NAD+ and bulk NAD-capped RNA during infection, we observe parallel decreases in both parameters over the course of infection. Furthermore, we characterize NudE.1, a T4 phage-encoded Nudix hydrolase previously shown to have in vitro NAD-RNA decapping activity. Together, our work presents the first time-resolved analysis of an RNA modification in a host-phage system, defining the landscape, dynamics, and turnover of NAD-capped RNAs during infection and providing a framework for future studies addressing their regulatory functions in phage biology.

microbiology↗

bioRxiv2024

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

microbiology↗