1,856 research outputs found
Characterisation of host growth after infection with a broad-range freshwater cyanopodophage
Freshwater cyanophages are poorly characterised in comparison to their marine counterparts, however, the level of genetic diversity that exists in freshwater cyanophage communities is likely to exceed that found in marine environments, due to the habitat heterogeneity within freshwater systems. Many cyanophages are specialists, infecting a single host species or strain; however, some are less fastidious and infect a number of different host genotypes within the same species or even hosts from different genera. Few instances of host growth characterisation after infection by broad host-range phages have been described. Here we provide an initial characterisation of interactions between a cyanophage isolated from a freshwater fishing lake in the south of England and its hosts. Designated ΦMHI42, the phage is able to infect isolates from two genera of freshwater cyanobacteria, Planktothrix and Microcystis. Transmission Electron Microscopy and Atomic Force Microscopy indicate that ΦMHI42 is a member of the Podoviridae, albeit with a larger than expected capsid. The kinetics of host growth after infection with ΦMHI42 differed across host genera, species and strains in a way that was not related to the growth rate of the uninfected host. To our knowledge, this is the first characterisation of the growth of cyanobacteria in the presence of a broad host-range freshwater cyanophage
Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage
Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites
The effect of phosphorus and nitrogen limitation on viral infection in microcystis aeruginosa NIES298 using the cyanophage Ma-LMM01
Throughout different times in freshwater cyanobacterial harmful algal blooms (cHAB), the availability of specific nutrients from the environment varies, causing fluctuations in the severity and species composition of a bloom. In addition to nutrient regulation of blooms, the cyanobacteria are subject to regulation by biotic factors, including phage infection. To address the potential role of cyanophages on the dominant species in most cHABs during nutrient-limited periods, we studied a specific host-phage system: Ma- LMM01 (phage) and Microcystis aeruginosa strain NIES298 (host), both of which originate from a eutrophic lake in Japan. The effect of phosphate and nitrogen limitation on phage and host replication was evaluated through modification of M. aeruginosa culture media. Growth of the host cells was monitored by culture light absorbance at 600 nm, while phage (genome) replication was quantified using real-time quantitative PCR (qPCR). Under phosphorus-limited conditions, Ma-LMM01 infected cells demonstrated a decrease in growth rate and carrying capacity compared to uninfected and infected nonlimited cultures. This relationship suggests that phage infection decreases M. aeruginosa growth to a greater degree under phosphorous stress than when the nutrient is readily available. In this model, these results indicate cyanophage replication may accelerate cHAB collapse under phosphorus-limiting conditions, and that increased concentrations of phosphorus may decrease the impact of cyanophage infections in the wild
Quantification of Toxin Biosynthesis Genes In Cyanobacteria and Dinoflagellates - Genetic Factors as Predictors of Toxin Production in the Environment
Harmful algal blooms (HABs) are events caused by the massive proliferation of microscopic, often photosynthetic organisms that inhabit both fresh and marine waters. Although HABs are essentially a natural phenomenon, they now cause worldwide concern. Recent anthropogenic effects, such as climate change and eutrophication via nutrient runoff, can be seen in their increased prevalence and severity. Cyanobacteria and dinoflagellates are often the causative organisms of HABs. In addition to adverse effects caused by the sheer biomass, certain species produce highly potent toxic compounds: hepatotoxic microcystins are produced exclusively by cyanobacteria and neurotoxic saxitoxins, also known as paralytic shellfish toxins (PSTs), by both cyanobacteria and dinoflagellates. Specific biosynthetic genes in the cyanobacterial genomes direct the production of microcystin and paralytic shellfish toxins. Recently also the first paralytic shellfish toxin gene sequences from dinoflagellate genomes have been elucidated.
The public health risks presented by HABs are evident, but the monitoring and prediction of toxic events is challenging. Characterization of the genetic background of toxin biosynthesis, including that of microcystins and paralytic shellfish toxins, has made it possible to develop highly sensitive molecular tools which have shown promise in the monitoring and study of potentially toxic microalgae. In this doctoral work, toxin-specific genes were targeted in the developed PCR and qPCR assays for the detection and quantification of potentially toxic cyanobacteria and dinoflagellates in the environment. The correlation between the copy numbers of the toxin biosynthesis genes and toxin production were investigated to assess whether the developed methods could be used to predict toxin concentrations.
The nature of the correlation between gene copy numbers and amount of toxin produced varied depending on the targeted gene and the producing organism. The combined mcyB copy numbers of three potentially microcystin-producing cyanobacterial genera showed significant positive correlation to the observed total toxin production. However, the presence of PST-specific sxtA, sxtG, and sxtB genes of cyanobacterial origin was found to be a poor predictor of toxin production in the studied area. Conversely, the dinoflagellate sxtA4 was a good qualitative indicator of a neurotoxic bloom both in the laboratory and in the field, and population densities reflected well the observed toxin concentrations. In conclusion, although the specificity of each potential targeted toxin biosynthesis gene must be assessed individually during method development, the results obtained in this doctoral study support the use of quantitative PCR -based approaches in the monitoring of toxic cyanobacteria and dinoflagellates.Siirretty Doriast
Development and Bias Assessment of a Method for Targeted Metagenomic Sequencing of Marine Cyanobacteria
Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms in oligotrophic waters and responsible for a significant percentage of the earth's primary production. Here we developed a method for metagenomic sequencing of sorted Prochlorococcus and Synechococcus populations using a transposon-based library preparation technique. First, we observed that the cell lysis technique and associated amount of input DNA had an important role in determining the DNA library quality. Second, we found that our transposon-based method provided a more even coverage distribution and matched more sequences of a reference genome than multiple displacement amplification, a commonly used method for metagenomic sequencing. We then demonstrated the method on Prochlorococcus and Synechococcus field populations from the Sargasso Sea and California Current isolated by flow cytometric sorting and found clear environmentally related differences in ecotype distributions and gene abundances. In addition, we saw a significant correspondence between metagenomic libraries sequenced with our technique and regular sequencing of bulk DNA. Our results show that this targeted method is a viable replacement for regular metagenomic approaches and will be useful for identifying the biogeography and genome content of specific marine cyanobacterial populations
CHARACTERISATION OF LATENT INFECTIONS lN AQUATIC CYANOBACTERIA AND MICROALGAE
Aquatic photosynthetic microorganisms were surveyed (algae and cyanobacteria) for
novel lysogenic/latent viruses and new methodology was established, using a variety of
techniques such as AFC, electron microscopy, and molecular tools. The first study
assessed Symbiodinium sp. cultures as a model system to investigate the induction of
potential latent vi ruses. The study of Symbiodinium sp. showed that ca. 37% of the
strains tested had a group of filamentous VLPs that is inducible by UV-C treatment.
Extrapolation of this virus-host interaction and its effects on zooxanthellae viability
provides a novel link to the impact of latent infection on symbiotic dinoflagellates of
cnidarians and the subsequent disruption of the reef ecosystems. The second study
examined the interaction of a freshwater cyanobacterium and its inducible VLPs. The
work carried out on Pseudanabaena, strain PPt10905 suggests that this freshwater
cyanobacterium harbours a prophage. An unusual interaction was observed in this
freshwater cyanobacterium, where the abundance of carboxysome-like particles
increased 10 times in heat-treated cultures. The cyanobacterium PPt10905, its inducible
VLPs and the co-occurring increase in carboxysomes could be a new mechanism in
which lysogeny benefits freshwater cyanobacteria, possibly increasing the host's
photosynthetic efficiency. Finally, the third study investigated the presence of latent
viruses in the Plymouth culture collection of marine algae. The characterisation and
isolation of inducible viruses from this algal culture collection has revealed much novel
information on the prevalence of latent viruses in algae. From the 30 algal species
examined in this study, over 35% appear to contain an inducible infectious agent. AFC
and TEM images have confirmed the presence of VLPs, and thin sections of UV-induced
cultures further supported the presence of VLPs in the UV-induced cultures.
This work's contribution increases the knowledge of latent and temperate viruses of
aquatic microbes, which are underrepresented in previous studies. Additionally, this
research established novel techniques for the study of unique biological interactions
between aquatic viruses and their hosts that will facilitate and improve subsequent
investigation of similar systems
The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms
Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate
Whole genome sequencing of cyanobacterium Nostoc sp. CCCryo 231-06 using microfluidic single cell technology
The Nostoc sp. strain CCCryo 231-06 is a cyanobacterial strain capable of surviving under extreme conditions and thus is of great interest for the astrobiology community. The knowledge of its complete genome sequence would serve as a guide for further studies. However, a major concern has been placed on the effects of contamination on the quality of sequencing data without a reference genome. Here, we report the use of microfluidic technology combined with single cell sequencing and de novo assembly to minimize the contamination and recover the complete genome of the Nostoc strain CCCryo 231-06 with high quality. 100% of the whole genome was recovered with all contaminants removed and a strongly supported phylogenetic tree. The data reported can be useful for comparative genomics for phylogenetic and taxonomic studies. The method used in this work can be applied to studies that require high-quality assemblies of genomes of unknown microorganisms
Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B.
UnlabelledNitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis.ImportanceBacterial nitric oxide (NO) signaling via heme-nitric oxide/oxygen binding (H-NOX) proteins regulates biofilm formation, playing an important role in protecting bacteria from oxidative stress and other environmental stresses. Biofilms are also an important part of symbiosis, allowing the organism to remain in a nutrient-rich environment. In this study, we show that in Silicibacter sp. strain TrichCH4B, NO mediates symbiosis with the alga Trichodesmium erythraeum, a major marine diazotroph. In addition, Silicibacter sp. TrichCH4B is the first characterized bacteria to harbor both the NOS and H-NOX proteins, making it uniquely capable of both synthesizing and sensing NO, analogous to mammalian NO signaling. Our study expands current understanding of the role of NO in bacterial signaling, providing a novel role for NO in bacterial communication and symbiosis
- …