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Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases. Editor's evaluation This important work substantially advances our understanding of the mechanisms phages use to evade host defenses. Specifically, the authors use computational and theoretical analyses of tRNA-encoding phages that infect several bacterial species to identify a novel, potential mechanism through which phage-encoded tRNAs help these phages evade tRNA cleavage that is induced as a host defense. Although the evidence supporting the conclusions is compelling, with multiple observations suggesting that the phage-encoded tRNAs have evolved to evade host-encoded tRNases, the conclusions would have been more strongly supported by providing an experimental test of the hypothesis., BN/Stan Brouns Lab

Serratia sp. ATCC 39006 is a Gram-negative bacterium that has been used to study the function of phage defences, such as CRISPR-Cas, and phage counter-defence mechanisms. To expand our phage collection to study the phage-host interaction with Serratia sp. ATCC 39006, we isolated the T4-like myovirus LC53 in Ōtepoti Dunedin, Aotearoa New Zealand. Morphological, phenotypic and genomic characterization revealed that LC53 is virulent and similar to other Serratia, Erwinia and Kosakonia phages belonging to the genus Winklervirus. Using a transposon mutant library, we identified the host ompW gene as essential for phage infection, suggesting that it encodes the phage receptor. The genome of LC53 encodes all the characteristic T4-like core proteins involved in phage DNA replication and generation of viral particles. Furthermore, our bioinformatic analysis suggests that the transcriptional organization of LC53 is similar to that of Escherichia coli phage T4. Importantly, LC53 encodes 18 tRNAs, which likely compensate for differences in GC content between phage and host genomes. Overall, this study describes a newly isolated phage infecting Serratia sp. ATCC 39006 that expands the diversity of phages available to study phage-host interactions., BN/Stan Brouns Lab

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we confirm that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo., Dynamics of Micro and Nano Systems, BN/Bionanoscience, BN/Chirlmin Joo Lab, BN/Dimphna Meijer Lab, BN/Cees Dekker Lab, BN/Stan Brouns Lab

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections., BN/Stan Brouns Lab

Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb-gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA-induced protease activity to gain viral immunity., Accepted Author Manuscript, BN/Stan Brouns Lab, BN/Technici en Analisten, BT/Biocatalysis

Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb-gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA-induced protease activity to gain viral immunity., Accepted Author Manuscript, BN/Stan Brouns Lab, BN/Technici en Analisten, BT/Biocatalysis

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections., BN/Stan Brouns Lab