Biology subjects

Olina, A.

Publications and source records attributed to Olina, A..

5 recordsLinked to original sources

bioRxiv2026

Loss of restriction-modification methyltransferases drives persistence to fluoroquinolones in Pseudomonas aeruginosa

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

microbiology↗

bioRxiv2026

Dual ATPase-activated DNA hemimethylation and cleavage by the MANTIS defence system

Bacterial defence systems are diverse and form major barriers to horizontal gene transfer and bacteriophage (phage) infection. Though restriction-modification systems were the first such defences discovered and remain the most widespread, the basis of self/non-self discrimination in many newly identified methylation-based systems remains unclear. Here, we define the mechanism of a widely distributed BREX-related defence family, previously termed Gao_RL, which we rename MANTIS (Methyltransferase, ATPases and Nuclease Targeting Inverted-repeat Sequences). Using phage infection and plasmid transformation assays in Pseudomonas aeruginosa, biochemical reconstitution, and structural biology, we show that MANTIS establishes self-identity through adenine hemimethylation of short asymmetric DNA sequences. Unexpectedly, a AAA+ ATPase (MtsA) and a co-regulator (MtsB) are both required to recruit and activate the methyltransferase MtsC, revealing a previously unknown ATP-dependent mode of epigenetic modification. DNA recognition by the MtsABC complex then triggers restriction by a dimeric helicase-nuclease effector (MtsD), with activity dependent on target-site architecture: inverted pairs of unmodified sites support robust restriction, whereas direct repeats or single sites are restricted less efficiently. Consistent with this model, virulent phages show strand-biased depletion of MANTIS target sites, indicating evolutionary pressure to evade restriction. Together, these findings reveal how dual ATPases couple self-modification to non-self restriction and provide a framework for understanding the expanding landscape of methylation-based defence systems.

molecular biology↗

bioRxiv2026

Engineering the Pseudomonas aeruginosa virus-isolation host viPAO1 reveals how host genetic barriers shape recoverable phage diversity

Cultured phage collections provide essential access to viral biology, but they capture only a small and biased fraction of natural phage diversity. Key barriers to diverse phage isolation are likely to include the prophages and defence systems present in the genomes of isolation hosts. Here, we show that removal of these host genetic barriers expands recoverable phage diversity in Pseudomonas aeruginosa. We constructed a series of progressively more permissive hosts derived from MPAO1 by sequentially deleting intracellular defence systems and resident prophages, culminating in viPAO1 (virus-isolation PAO1). Sequential removal of these barriers increased phage susceptibility, with Gabija and type I restriction-modification acting as major barriers to phage infection. In comparative isolation experiments, removal of intracellular barriers produced the largest increase in phage recovery, while additional receptor modification altered the composition of recovered phage subsets. viPAO1 also substantially increased recovery of phage contigs from environmental samples. Scaling this approach across 1090 clinical isolates enabled recovery of a curated collection of 69 individually purified temperate P. aeruginosa phages spanning 24 predicted genera, including 15 putative novel genera. Together, these findings demonstrate that removal of host genetic barriers to infection enhances recoverable phage diversity and establish permissive host engineering as a practical strategy for uncovering under-sampled regions of cultured phage diversity.

microbiology↗

bioRxiv2022

Bacterial Argonaute proteins aid cell division in the presence of topoisomerase inhibitors in Escherichia coli

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix roseae (LrAgo) act as DNA-guided DNA nucleases in Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin. Both pAgos are preferentially loaded with small DNA guides derived from the sites of replication termination. The amount of pAgo-associated small DNAs (smDNAs) from the termination sites is increased in the presence ciprofloxacin, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin also enhances asymmetry in the distribution of smDNAs around Chi-sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases help to complete replication of chromosomal DNA by targeting the sites of termination, and may switch their functional activities when expressed in different host species.

molecular biology↗

bioRxiv2022

Specific targeting of plasmids with Argonaute enables genome editing

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. Recent studies showed that pAgos can bind small single-stranded guide DNAs (gDNAs) to recognize and cleave complementary DNA in vitro. In vivo pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we reveal that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA cleavage and engineering in bacteria. CbAgo-dependent targeting of genomic loci with plasmid-derived gDNAs promotes recombination between plasmid and chromosomal DNA. Efficient genome cleavage and recombineering depends on the catalytic activity of CbAgo, its interactions with gDNAs, and the extent of homology between plasmid and chromosomal sequences. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing. One-Sentence SummaryProkaryotic Argonaute nuclease induces DNA interference between plasmid and chromosomal DNA to promote genome recombineering.

molecular biology↗