Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria. IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.Peer reviewe

Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions.

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers

Salt resistance mechanism of halotolerant / halophilic prokaryotic DNases and halotolerance induction for bovine DNaseI

Analysis of the sequence data revealed that many prokaryotic DNaseI-like nucleases from halotolerant / halophilic species are multi domain proteins. This fact led to a hypothesis that in some cases a fusion of an additional domain to the DNase domain was the key factor in evolution that enabled the activity of prokaryotic DNases at high ionic strength. In this study the hypothesis was experimentally proved by analysing halo- tolerance of one DNase from Thioalkalivibrio sp. K90mix (DNaseTA) and its mutants. DNaseTA is comprised of two domains: one domain is DNaseI-like and the other is a DNA-binding domain comprising two HhH (helix-hairpin- helix) motifs. It was decided to mimic in vitro the evolutionary step that created the natural fusion. The research revealed that this domain originated from ComEA/ComE proteins (DNA receptor of bacterial competence system) and through the course of evolution was fused with the DNaseI-like domain. In this study the domain organization of DNaseTA was mimicked by creating two fusion proteins comprising bovine DNaseI and a DNA-binding domain. Both fusions with additional DNA binding domains were demonstrated to be more salt tolerant than bovine DNaseI, albeit to different extent. Molecular modelling data suggested that differences in tolerance for high ionic strength between the two created DNaseI fusions could be due to differences in hydrogen bonding between the fused domains and DNA and different ability to transfer monovalent cations to solvent during DNA-protein complex formation

Prokariotinių DNazės I homologų pakantumo druskingumui / halofiliškumo mechanizmai bei jaučio DNazės I atsparumo joninei jėgai didinimas

Analysis of the sequence data revealed that many prokaryotic DNaseI-like nucleases from halotolerant / halophilic species are multi domain proteins. This fact led to a hypothesis that in some cases a fusion of an additional domain to the DNase domain was the key factor in evolution that enabled the activity of prokaryotic DNases at high ionic strength. In this study the hypothesis was experimentally proved by analysing halo- tolerance of one DNase from Thioalkalivibrio sp. K90mix (DNaseTA) and its mutants. DNaseTA is comprised of two domains: one domain is DNaseI-like and the other is a DNA-binding domain comprising two HhH (helix-hairpin- helix) motifs. It was decided to mimic in vitro the evolutionary step that created the natural fusion. The research revealed that this domain originated from ComEA/ComE proteins (DNA receptor of bacterial competence system) and through the course of evolution was fused with the DNaseI-like domain. In this study the domain organization of DNaseTA was mimicked by creating two fusion proteins comprising bovine DNaseI and a DNA-binding domain. Both fusions with additional DNA binding domains were demonstrated to be more salt tolerant than bovine DNaseI, albeit to different extent. Molecular modelling data suggested that differences in tolerance for high ionic strength between the two created DNaseI fusions could be due to differences in hydrogen bonding between the fused domains and DNA and different ability to transfer monovalent cations to solvent during DNA-protein complex formation

Enhancement of DNaseI salt tolerance by mimicking the domain structure of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix

In our previous work we showed that DNaseI-like protein from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix retained its activity at salt concentrations as high as 4 M NaCl and the key factor allowing this was the C-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs. The further investigations revealed that this domain originated from proteins related to bacterial competence ComEA/ComE proteins. It is likely that in the course of evolution the DNA-binding domain from these proteins was fused to a metallo-β-lactamase superfamily domain. Very likely such domain organization having proteins subsequently “donated” the DNA-binding domain to bacterial DNases. In this study we have mimicked this evolutionary step by fusing bovine DNaseI and DNA-binding domains. We have created two fusions: one harboring the DNA-binding domain of DNaseI-like protein from Thioalkalivibrio sp. K90mix and the second one harboring the DNA-binding domain of bacterial competence protein ComEA from Bacillus subtilis. Both domains enhanced salt tolerance of DNaseI, albeit to different extent. Molecular modeling revealed the essential differences between their interaction with DNA shedding some light on the differences in salt tolerance. In this study we have enhanced salt tolerance of bovine DNaseI; thus, we successfully mimicked the Nature’s evolutionary engineering that created the extremely halotolerant bacterial DNase. We have demonstrated that the newly engineered DNaseI variants can be successfully used in applications where activity of the wild type bovine DNaseI is impeded by buffers used

Superposition of structural models of two (HhH)₂ domains from <i>Thioalkalivibrio sp. K90mix</i> (blue colour) and <i>Bacillus subtilis</i> (green colour) and corresponding sequence alignment.

<p>DNA phosphate contacting positive residues are indicated by red colour in the sequence alignment and by stick representations in the structural alignment. Yellow colour indicates part of the domain from <i>Bacillus subtilis</i>, which has no corresponding residues in the domain from <i>Thioalkalivibrio sp. K90mix</i>. Approximate position of DNA is indicated by transparent sticks based on structure PDBID: 3E0D after superimposition with the domains.</p

Domain organization of DNase from Thioalkalivibrio sp. provides insights into retention of activity in high salt environments

Our study indicates that DNA binding domains are common in many halophilic or halotolerant bacterial DNases and they are potential activators of enzymatic activity at high ionic strength. Usually, proteins adapt to high ionic strength by increasing the number of negatively charged residues on the surface. However, in DNases such adaptation would hinder the binding to negatively charged DNA, a step critical for catalysis. In our study we demonstrate how evolution has solved this dilemma by engaging the DNA binding domain. We propose a mechanism, which enables the enzyme activity at salt concentrations as high as 4 M of sodium chloride, based on collected experimental data and domain structure analysis of a secreted bacterial DNase from the extremely halotolerant bacterium Thioalkalivibrio sp. K90mix. The enzyme harbors two domains: an N-terminal domain, that exhibits DNase activity, and a C-terminal domain, comprising a duplicate DNA binding helix-hairpin-helix motif. Here we present experimental data demonstrating that the C-terminal domain is responsible for the enzyme’s resistance to high ionic strength

Yield of DNaseI fusions and general characterisation of used protein samples.

<p>DNaseDT denotes the fusion with the (HhH)₂ domain of DNase from <i>Thioalkalivibrio sp. K90mix</i>. DNaseBS—the fusion with homologous domain of ComEA protein from <i>Bacillus subtilis</i>.</p

Changes in polar electrostatic solvation energy upon complex formation at different NaCl concentrations.

<p>Two domains were modeled: one from an extremely salt tolerant bacterium <i>Thioalkalivibrio sp. K90mix</i> (DT), the other one from <i>Bacillus subtilis</i>(BS).</p

Electrostatic surface potential of DNA-binding surface and changes in local ion concentration upon binding to DNA.

<p>Electrostatic potential of DNA-binding surface of the domain from <i>Bacillus subtilis</i> is shown on the upper-left (A) and corresponding surface of the domain from <i>Thioalkalivibrio sp. K90mix</i> is given on the upper-right (B). The range from −1.5 kT/e in red to +1.5 kT/e in blue was chosen for surface colouring. The surface is semi transparent and stick representations of the DNA phosphates contacting residues are visible: lysines—in case of <i>Bacillus subtilis</i> domain and arginines in case of <i>Thioalkalivibrio sp. K90 mix</i> domain. The changes in local ion concentrations upon formation of a complex with DNA by the domain from <i>Bacillus subtilis</i> are depicted in the lower-left (C), the corresponding changes in the case of the domain from <i>Thioalkalivibrio sp. K90mix</i> are depicted in the lower-right (D). The DNA phosphates interacting residues are depicted by white sticks. The isocountour surfaces indicate the changes, which occur in the presence of 0.4 M NaCl. Four isocountour surfaces are visualized simultaneously. The deep blue resembles changes in local ion concentration equivalent to −3 M, the lighter blue resembles changes equivalent to −2 M. Similarly deep red indicates changes equivalent to +3 M and the lighter red indicates changes equivalent to +2 M. Isosurfaces equivalent to +2/-2 M overlaps corresponding isosurfaces which resemble changes equivalent to +3/-3 M.</p