RESUMO
Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.
Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Ribonucleases/genética , Sistemas Toxina-Antitoxina/genética , Monofosfato de Adenosina/genética , Antídotos/química , Antitoxinas/metabolismo , Aphanizomenon/química , Aphanizomenon/genética , Sistemas CRISPR-Cas/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ribonucleases/metabolismo , Tirosina/genéticaRESUMO
Studies of the diversity and distribution of freshwater cyanophages are generally limited to the small geographical areas, in many cases including only one or few lakes. Data from dozens of various lakes distributed at a larger distance are necessary to understand their spatial distribution and sensitivity to biotic and abiotic factors. Thus, the objective of this study was to analyze the diversity and distribution of cyanophages within the infected cells using marker genes (psbA, nblA, and g91) in 21 Polish and Lithuanian lakes. Physicochemical factors that might be related to them were also analyzed. The results demonstrated that genetic markers representing cyanophages were observed in most lakes studied. The frequently detected gene was psbA with 88% of cyanophage-positive samples, while nblA and g91 were found in approximately 50% of lakes. The DNA sequence analyses for each gene demonstrated low variability between them, although the psbA sequences branched within the larger cluster of marine Synechoccocuss counterparts. The principal component analysis allowed to identify significant variation between the lakes that presented high and low cyanobacterial biomass. The lakes with high cyanobacterial biomass were further separated by country and the different diversity of cyanobacteria species, particularly Planktothrix agardhii, was dominant in the Polish lakes and Planktolyngbya limnetica in the Lithuanian lakes. The total phosphorous and the presence of cyanophage genes psbA and nblA were the most important factors that allowed differentiation for the Polish lakes, while the pH and the genes g91 and nblA for the Lithuanian lakes.
Assuntos
Bacteriófagos , Lagos , Bacteriófagos/genética , Marcadores Genéticos , Filogenia , Reação em Cadeia da PolimeraseRESUMO
In the photic zone of aquatic ecosystems, microorganisms with different metabolisms and their viruses form complex interactions and food webs. Within these interactions, phototrophic microorganisms such as eukaryotic microalgae and cyanobacteria interact directly with sunlight, and thereby generate circadian rhythms. Diel cycling originally generated in microbial phototrophs is directly transmitted toward heterotrophic microorganisms utilizing the photosynthetic products as they are excreted or exuded. Such diel cycling seems to be indirectly propagated toward heterotrophs as a result of complex biotic interactions. For example, cell death of phototrophic microorganisms induced by viral lysis and protistan grazing provides additional resources of dissolved organic matter to the microbial community, and so generates diel cycling in other heterotrophs with different nutrient dependencies. Likewise, differences in the diel transmitting pathway via complex interactions among heterotrophs, and between heterotrophs and their viruses, may also generate higher variation and time lag diel rhythms in different heterotrophic taxa. Thus, sunlight and photosynthesis not only contribute energy and carbon supply, but also directly or indirectly control diel cycling of the microbial community through complex interactions in the photic zone of aquatic ecosystems.
Assuntos
Organismos Aquáticos/fisiologia , Microbiota/fisiologia , Periodicidade , Organismos Aquáticos/metabolismo , Carbono/metabolismo , Metabolismo Energético , Cadeia Alimentar , Interações Microbianas , Modelos Biológicos , Fotossíntese , Luz SolarRESUMO
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.
Assuntos
Aphanizomenon/virologia , Genoma Viral/genética , Siphoviridae/genética , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Viral/análise , Lituânia , Filogenia , Siphoviridae/classificaçãoRESUMO
Cyanobacteria play a significant role in ecosystem functioning as photosynthetic and CO2 fixing microorganisms. Whether and to what extent cyanophages alter these carbon and energy cycles in their cyanobacterial hosts is still poorly understood. In this study, we investigated changes in photosynthetic activity (PSII), expression of genes associated with the light phase of photosynthesis (psbA, petA, ndhK) and carbon metabolism (rbcL, zwf) as well as intracellular ATP and NADHP concentrations in freshwater bloom-forming filamentous cyanobacterium Aphanizomenon flos-aquae infected by cyanophage vB_AphaS-CL131. We found that PSII activity and expression level of rbcL genes, indicating potential for CO2 fixation, had decreased in response to cyanophage adsorption and DNA injection. During the period of viral DNA replication and assembly, PSII performance and gene expression remained at this decreased level and did not change significantly, indicating lack of transcriptional shutdown by the cyanophage. Combined, these observations suggest that although there is little to no interference between cyanophage DNA replication, host transcription and cellular metabolism, A. flos-aquae underwent a physiological state-shift toward lower efficiency of carbon and energy cycling. This further suggest potential cascading effect for co-occurring non-infected members of the microbial community.
Assuntos
Dióxido de Carbono , Cianobactérias , Aphanizomenon , Carbono , Replicação do DNA , DNA Viral , Ecossistema , Água Doce , Fotossíntese , Replicação ViralRESUMO
Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus-host interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.
Assuntos
Bacteriófagos/genética , Chlorobium/virologia , Lagos/microbiologia , Viroma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/isolamento & purificação , Bacteriófagos/patogenicidade , Sulfato de Cálcio/análise , Sulfato de Cálcio/metabolismo , Chlorobium/genética , Chlorobium/metabolismo , Genômica/métodos , Interações Hospedeiro-Patógeno , Lagos/química , Lagos/virologia , Metagenoma , Análise de Célula Única/métodos , Enxofre/metabolismoRESUMO
Freshwater bloom-forming cyanobacteria densely grow in the aquatic environments, leading to an increase in the viral-contact rate. They possess numerous antiviral genes, as well as cell differentiation- and physiological performance-related genes, owing to genome expansion. Their genomic features and unique lifestyles suggest that they coexist with cyanoviruses in ways different from marine cyanobacteria. Furthermore, genome contents of isolated freshwater bloom-forming cyanobacterial viruses have little in common with those of marine cyanoviruses studied to date. They lack the marine cyanoviral hallmark genes that sustain photosynthetic activity and redirect host metabolism to viral reproduction; therefore, they are predicted to share metabolisms and precursor pools with host cyanobacteria to ensure efficient viral reproduction and avoid nutrient deficiencies and antiviral response. Additionally, cyanovirus-cyanobacteria coexistence strategies may change as bloom density increases. Diverse genotypic populations of cyanoviruses and hosts coexist and fluctuate under high viral-contact rate conditions, leading to their rapid coevolution through antiviral responses. The ancestral and newly evolved genotypes coexist, thereby expanding the diversity levels of host and viral populations. Bottleneck events occurring due to season-related decreases in bloom-forming species abundance provide each genotype within cyanobacterial population an equal chance to increase in prevalence during the next bloom and enhance further diversification.
Assuntos
Bacteriófagos/genética , Cianobactérias/virologia , Água Doce/microbiologia , Água Doce/virologia , Vírus/genética , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Bacteriófagos/metabolismo , Cianobactérias/classificação , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Genoma Viral , Genômica , Filogenia , Vírus/classificação , Vírus/isolamento & purificação , Vírus/metabolismoRESUMO
In this study, we present the genomic characterization of the temperate bacteriophage vB_BceS_KLEB30-3S (KLEB30-3S), which was induced from Bacillus cereus strain KR3M-30, isolated from a gypsum karst lake ecosystem in Lithuania. The 37,134-bp genome of KLEB30-3S contains 58 predicted protein-encoding genes and no tRNA genes.
RESUMO
Viruses can significantly influence cyanobacteria population dynamics and activity, and through this the biogeochemical cycling of major nutrients. However, surprisingly little attention has been given to understand how viral infections alter the ability of diazotrophic cyanobacteria for atmospheric nitrogen fixation and its release to the environment. This study addressed the importance of cyanophages for net 15N2 assimilation rate, expression of nitrogenase reductase gene (nifH) and changes in nitrogen enrichment (15N/14N) in the diazotrophic cyanobacterium Aphanizomenon flos-aquae during infection by the cyanophage vB_AphaS-CL131. We found that while the growth of A. flos-aquae was inhibited by cyanophage addition (decreased from 0.02 h-1 to 0.002 h-1), there were no significant differences in nitrogen fixation rates (control: 22.7 × 10-7 nmol N heterocyte-1; infected: 23.9 × 10-7 nmol N heterocyte-1) and nifH expression level (control: 0.6-1.6 transcripts heterocyte-1; infected: 0.7-1.1 transcripts heterocyte-1) between the infected and control A. flos-aquae cultures. This implies that cyanophage genome replication and progeny production within the vegetative cells does not interfere with the N2 fixation reactions in the heterocytes of these cyanobacteria. However, higher 15N enrichment at the poles of heterocytes of the infected A. flos-aquae, revealed by NanoSIMS analysis indicates the accumulation of fixed nitrogen in response to cyanophage addition. This suggests reduced nitrogen transport to vegetative cells and the alterations in the flow of fixed nitrogen within the filaments. In addition, we found that cyanophage lysis resulted in a substantial release of ammonium into culture medium. Cyanophage infection seems to substantially redirect N flow from cyanobacterial biomass to the production of N storage compounds and N release.
RESUMO
The effect of cyanophage infection and lysis on the dynamics of the hepatotoxin nodularin (NOD) and other non-ribosomal peptides (NRPs) produced by cyanobacteria is poorly understood. In this study, changes in concentration of NOD and other NRPs during cyanophage infection of the filamentous cyanobacteria Nodularia spumigena were assessed using incubation experiments. Viral infection and lysis were associated with a significant reduction (93% at the 96 h post infection) of N. spumigena cell density. While no correlation between N. spumigena abundance and total concentration of NOD (ng mL-1) within the infected cells was observed, cellular NOD quota (ng cell-1) gradually increased in the remaining cyanophage resistant N. spumigena subpopulation. Lysis of N. spumigena cells resulted in a substantial increase (>57 times) of dissolved NOD concentration in the culture medium. The relative concentration of other cyclic (anabaenopeptins) and linear (aeruginosins, spumigins) NRPs produced by N. spumigena also increased in response to cyanophage addition. This study highlights the importance of cyanophage infection on the population toxicity of filamentous cyanobacteria and demonstrates a significant contribution of virus-mediated cell lysis on the conversion of NOD from the particulate to dissolved phase.
Assuntos
Bacteriófagos/fisiologia , Cianobactérias/metabolismo , Cianobactérias/virologia , Peptídeos Cíclicos/metabolismo , Biodegradação Ambiental , Cromatografia Líquida , Monitoramento Ambiental , Peptídeos/metabolismo , Densidade Demográfica , Espectrometria de Massas em TandemRESUMO
In order to better understand the relationships among current Nostocales cyanobacterial blooms, eight genomes were sequenced from cultured isolates or from environmental metagenomes of recent planktonic Nostocales blooms. Phylogenomic analysis of publicly available sequences placed the new genomes among a group of 15 genomes from four continents in a distinct ADA clade (Anabaena/Dolichospermum/Aphanizomenon) within the Nostocales. This clade contains four species-level groups, two of which include members with both Anabaena-like and Aphanizomenon flos-aquae-like morphology. The genomes contain many repetitive genetic elements and a sizable pangenome, in which ABC-type transporters are highly represented. Alongside common core genes for photosynthesis, the differentiation of N2-fixing heterocysts, and the uptake and incorporation of the major nutrients P, N and S, we identified several gene pathways in the pangenome that may contribute to niche partitioning. Genes for problematic secondary metabolites-cyanotoxins and taste-and-odor compounds-were sporadically present, as were other polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters. By contrast, genes predicted to encode the ribosomally generated bacteriocin peptides were found in all genomes.
Assuntos
Cianobactérias/classificação , Genoma Bacteriano , Proteínas de Bactérias/análise , Cianobactérias/genética , Proliferação Nociva de Algas , FilogeniaRESUMO
Sustainable and effective water management plans must have a reliable risk assessment strategies for harmful cyanobacterial blooms (HABs) that would enable timely decisions to be made, thus avoiding the trespassing of ecological thresholds, leading to the collapse of ecosystem structure and function. Such strategies are usually based on cyanobacterial biomass and/or on the monitoring of known toxins, which may, however, in many cases, under- or over-represent the actual toxicity of the HAB. Therefore, in this study, by the application of growth-inhibition assays using different bacteria, algae, zooplankton and fish species, we assessed the toxicological potential of two cyanobacterial blooms that differed in total cyanobacterial biomass, species composition and cyanopeptide profiles. We demonstrated that neither cyanobacterial community composition nor its relative abundance, nor indeed concentrations of known toxins reflected the potential risk of HAB based on growth-inhibition assays. We discuss our findings in the context of food-web dynamics and ecosystem management, and suggest that toxicological tests should constitute a key element in the routine monitoring of water bodies so as to prevent under-/over-estimation of potential HAB risk for both ecosystem and public health.
Assuntos
Conservação dos Recursos Naturais , Cianobactérias/crescimento & desenvolvimento , Eutrofização , Cadeia Alimentar , Monitoramento AmbientalRESUMO
Vb-AphaS-CL131 is a novel cyanosiphovirus that infects harmful Aphanizomenon flos-aquae. This cyanophage has an isometric head, 97 nm in diameter and a long, flexible non-contractile tail, 361 nm long. With a genome size of ~120 kb, it is the second largest cyanosiphovirus isolated to date. The latent period was estimated to be ~36 h and a single infected cell produces, on average, 218 infectious cyanophages. Cyanophage infection significantly suppresses host biomass production and alters population phenotype.
Assuntos
Aphanizomenon/virologia , Bacteriófagos/genética , Bacteriófagos/fisiologia , Aphanizomenon/isolamento & purificação , Estuários , Microbiologia da ÁguaRESUMO
We report here the de novo genome assembly of a cyanobacterium, Aphanizomenon flos-aquae strain 2012/KM1/D3, a harmful bloom-forming species in temperate aquatic ecosystems. The genome is 5.7 Mb with a G+C content of 38.2%, and it is enriched mostly with genes involved in amino acid and carbohydrate metabolism.
RESUMO
Concentration of toxic cyanobacteria blooms on the downwind shore of high recreational amenity water bodies with largely increases the risk of exposure to cyanotoxins. In this study analysis of phytoplankton structure, cyanotoxin composition and concentration was performed on cyanobacteria scum material, high- and low-density bloom samples in the Curonian Lagoon. We found that the concentration of cyanotoxins in the scum material increased from â¼30 to â¼300-fold compared to bloom samples. In Microcystis aeruginosa dominated samples microcystin-LR was present at the highest concentration, while the dominance of Planktothrix agardhii was associated with the occurrence of dmMC-RR as the major microcystin variant. The toxicological potential of cyanobacterial scums in the Curonian Lagoon is emphasized, and management by removal of these scums is proposed.