ABSTRACT
Cyanobacteria are oxygen-evolving photosynthetic bacteria. Established genetic manipulation methods and recently developed gene-regulation tools have enabled the photosynthetic conversion of carbon dioxide to biofuels and valuable chemicals in cyanobacteria, especially in unicellular cyanobacteria. However, the oxygen sensitivity of enzyme(s) introduced into cyanobacteria hampers productivity in some cases. Anabaena sp. PCC 7120 is a filamentous cyanobacterium consisting of a few hundred of vegetative cells, which perform oxygenic photosynthesis. Upon nitrogen deprivation, heterocysts, which are specialized cells for nitrogen fixation, are differentiated from vegetative cells at semiregular intervals. The micro-oxic environment within heterocysts protects oxygen-labile nitrogenase from oxygen. This study aimed to repurpose the heterocyst as a host for the production of chemicals with oxygen-sensitive enzymes under photosynthetic conditions. Herein, Anabaena strains expressing enzymes of 1-butanol synthetic pathway from the anaerobe Clostridium acetobutylicum within heterocysts were created. A strain that expressed a highly oxygen-sensitive Bcd/EtfAB complex produced 1-butanol even under photosynthetic conditions. Furthermore, the 1-butanol production per heterocyst cell of a butanol-producing Anabaena strain was fivefold higher than that per cell of unicellular cyanobacterium with the same set of 1-butanol synthetic pathway genes. Thus, our study showed the usefulness of Anabaena heterocysts as a chassis for anaerobic production driven by oxygen-evolving photosynthesis.
Subject(s)
Anabaena/metabolism , Butanols/metabolism , Metabolic Engineering/methods , Oxygen/metabolism , Photosynthesis/physiology , Anabaena/classification , Anabaena/genetics , Anaerobiosis , Bioreactors/microbiology , Clostridium acetobutylicum/enzymology , Clostridium acetobutylicum/geneticsABSTRACT
One simple model to explain biological pattern postulates the existence of a stationary regulator of differentiation that positively affects its own expression, coupled with a diffusible suppressor of differentiation that inhibits the regulator's expression. The first has been identified in the filamentous, heterocyst-forming cyanobacterium, Anabaena PCC 7120 as the transcriptional regulator, HetR and the second as the small protein, PatS, which contains a critical RGSGR motif that binds to HetR. HetR is present in almost all filamentous cyanobacteria, but only a subset of heterocyst-forming strains carry proteins similar to PatS. We identified a third protein, PatX that also carries the RGSGR motif and is coextensive with HetR. Amino acid sequences of PatX contain two conserved regions: the RGSGR motif and a hydrophobic N-terminus. Within 69 nt upstream from all instances of the gene is a DIF1 motif correlated in Anabaena with promoter induction in developing heterocysts, preceded in heterocyst-forming strains by an apparent NtcA-binding site, associated with regulation by nitrogen-status. Consistent with a role in the simple model, PatX is expressed dependent on HetR and acts to inhibit differentiation. The acquisition of the PatX/HetR pair preceded the appearance of both PatS and heterocysts, dating back to the beginnings of multicellularity.
Subject(s)
Anabaena , Bacterial Proteins/genetics , Peptides/genetics , Anabaena/classification , Anabaena/genetics , Gene Expression Regulation, Bacterial , Gene Regulatory Networks , Promoter Regions, GeneticABSTRACT
BACKGROUND: Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. RESULTS: The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. CONCLUSION: Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.
Subject(s)
Anabaena/genetics , Anabaena/metabolism , Genome, Bacterial , Genomics , Tropanes/metabolism , Anabaena/classification , Anabaena/isolation & purification , Computational Biology/methods , Cyanobacteria Toxins , Energy Metabolism , Genomics/methods , High-Throughput Nucleotide Sequencing , Metabolic Networks and Pathways , Metabolome , Metabolomics/methods , Molecular Sequence Annotation , Phylogeny , Promoter Regions, Genetic , Synteny , Tandem Repeat SequencesABSTRACT
The new genus name Dolichospermum, for most of the planktonic former members of the genus Anabaena, is one of the most ubiquitous bloom-forming cyanobacterial genera. Its dominance and persistence have increased in recent years, due to eutrophication from anthropogenic activities and global climate change. Blooms of Dolichospermum species, with their production of secondary metabolites that commonly include toxins, present a worldwide threat to environmental and public health. In this review, recent advances of the genus Dolichospermum are summarized, including taxonomy, genetics, bloom occurrence, and production of toxin and taste-and-odor compounds. The recent and continuing acquisition of genome sequences is ushering in new methods for monitoring and understanding the factors regulating bloom dynamics.
Subject(s)
Anabaena/classification , Anabaena/physiology , Bacterial Toxins/metabolism , Biodiversity , Harmful Algal Bloom/physiology , Anabaena/genetics , Anabaena/metabolism , Climate Change , Environmental Monitoring , EutrophicationABSTRACT
The factors and processes driving cyanobacterial blooms in eutrophic freshwater ecosystems have been extensively studied in the past decade. A growing number of these studies concern the direct or indirect interactions between cyanobacteria and heterotrophic bacteria. The presence of bacteria that are directly attached or immediately adjacent to cyanobacterial cells suggests that intense nutrient exchanges occur between these microorganisms. In order to determine if there is a specific association between cyanobacteria and bacteria, we compared the bacterial community composition during two cyanobacteria blooms of Anabaena (filamentous and N2-fixing) and Microcystis (colonial and non-N2 fixing) that occurred successively within the same lake. Using high-throughput sequencing, we revealed a clear distinction between associated and free-living communities and between cyanobacterial genera. The interactions between cyanobacteria and bacteria appeared to be based on dissolved organic matter degradation and on N recycling, both for N2-fixing and non N2-fixing cyanobacteria. Thus, the genus and potentially the species of cyanobacteria and its metabolic capacities appeared to select for the bacterial community in the phycosphere.
Subject(s)
Anabaena/genetics , Microcystis/genetics , Phylogeny , Phytoplankton/genetics , RNA, Ribosomal, 16S/genetics , Anabaena/classification , Biodiversity , Ecosystem , Eutrophication , Fresh Water , High-Throughput Nucleotide Sequencing , Microcystis/classification , Nitrogen/metabolism , Nitrogen Fixation , Phytoplankton/classificationABSTRACT
Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters--such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)--to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.
Subject(s)
Bacterial Toxins/metabolism , Harmful Algal Bloom , Microcystins/genetics , Microcystis/classification , Microcystis/genetics , Anabaena/classification , Anabaena/genetics , Anabaena/pathogenicity , Biomass , DNA, Bacterial/genetics , Lakes/microbiology , Microcystins/metabolism , Microcystis/pathogenicity , Polymerase Chain Reaction , WisconsinABSTRACT
Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.
Subject(s)
Antifungal Agents/isolation & purification , Aspergillus flavus/drug effects , Candida albicans/drug effects , Cyanobacteria/chemistry , Drug Discovery , Anabaena/chemistry , Anabaena/classification , Anabaena/growth & development , Anabaena/isolation & purification , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Aspergillus flavus/growth & development , Brazil , Candida albicans/growth & development , Cyanobacteria/classification , Cyanobacteria/growth & development , Cyanobacteria/isolation & purification , Czech Republic , Finland , Fresh Water/microbiology , Glycolipids/chemistry , Glycolipids/isolation & purification , Glycolipids/pharmacology , Lipopeptides/chemistry , Lipopeptides/isolation & purification , Lipopeptides/pharmacology , Molecular Structure , Molecular Typing , Nostoc/chemistry , Nostoc/classification , Nostoc/growth & development , Nostoc/isolation & purification , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Phylogeny , Pyrans/chemistry , Pyrans/isolation & purification , Pyrans/pharmacology , Saline Waters , Species SpecificityABSTRACT
Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⻹ to 4.92 µg·mg⻹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.
Subject(s)
Alkaloids/biosynthesis , Anabaena/metabolism , Aphanizomenon/metabolism , Bacterial Toxins/biosynthesis , Uracil/analogs & derivatives , Water Microbiology , Water Pollution, Chemical , Alkaloids/analysis , Alkaloids/chemistry , Alkaloids/isolation & purification , Anabaena/classification , Anabaena/growth & development , Anabaena/isolation & purification , Aphanizomenon/classification , Aphanizomenon/growth & development , Aphanizomenon/isolation & purification , Bacterial Toxins/analysis , Bacterial Toxins/chemistry , Bacterial Toxins/isolation & purification , Chromatography, High Pressure Liquid , Cyanobacteria Toxins , Lakes/microbiology , Molecular Typing , Seasons , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Turkey , Uracil/analysis , Uracil/biosynthesis , Uracil/chemistry , Uracil/isolation & purification , Water QualityABSTRACT
The symbiosis Azolla-Anabaena azollae, with a worldwide distribution in pantropical and temperate regions, is one of the most studied, because of its potential application as a biofertilizer, especially in rice fields, but also as an animal food and in phytoremediation. The cyanobiont is a filamentous, heterocystic cyanobacterium that inhabits the foliar cavities of the pteridophyte and the indusium on the megasporocarp (female reproductive structure). The classification and phylogeny of the cyanobiont is very controversial: from its morphology, it has been named Nostoc azollae, Anabaena azollae, Anabaena variabilis status azollae and recently Trichormus azollae, but, from its 16S rRNA gene sequence, it has been assigned to Nostoc and/or Anabaena, and from its phycocyanin gene sequence, it has been assigned as non-Nostoc and non-Anabaena. The literature also points to a possible co-evolution between the cyanobiont and the Azolla host, since dendrograms and phylogenetic trees of fatty acids, short tandemly repeated repetitive (STRR) analysis and restriction fragment length polymorphism (RFLP) analysis of nif genes and the 16S rRNA gene give a two-cluster association that matches the two-section ranking of the host (Azolla). Another controversy surrounds the possible existence of more than one genus or more than one species strain. The use of freshly isolated or cultured cyanobionts is an additional problem, since their morphology and protein profiles are different. This review gives an overview of how morphological, chemical and genetic analyses influence the classification and phylogeny of the cyanobiont and future research.
Subject(s)
Anabaena/classification , Phylogeny , Symbiosis , Anabaena/genetics , Biological Evolution , DNA, Bacterial/genetics , Ferns/microbiology , Phycocyanin/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes.
Subject(s)
Anabaena/genetics , Anabaena/pathogenicity , Bacterial Proteins/analysis , Gene Expression Regulation, Bacterial , Saxitoxin/biosynthesis , Anabaena/classification , Anabaena/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Ecotype , Ketoglutaric Acids/metabolism , Mass Spectrometry/methods , Molecular Sequence Annotation , Nitrogen/metabolism , Peptides/analysis , Photosynthesis/genetics , Phylogeny , Proteomics , Staining and Labeling/methods , VirulenceABSTRACT
In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation.
Subject(s)
Anabaena/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Regulon/physiology , Anabaena/classification , Anabaena/genetics , Bacterial Proteins/genetics , Binding Sites , Plasmids , Protein Binding , Transcription, GeneticABSTRACT
The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains - A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.
Subject(s)
Anabaena/drug effects , Fungi/growth & development , Pest Control, Biological , Phosphorus/pharmacology , Plant Diseases/microbiology , Anabaena/classification , Anabaena/enzymology , Anabaena/growth & development , Antibiosis , Cellulase/metabolism , Chlorophyll/metabolism , Fungi/pathogenicity , Fusarium/growth & development , Fusarium/pathogenicity , Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Microbial Sensitivity Tests , Pythium/growth & development , Pythium/pathogenicity , Rhizoctonia/growth & development , Rhizoctonia/pathogenicityABSTRACT
Blooms of toxic cyanobacteria, associated with illness and mortality in humans and animals, are becoming increasingly common worldwide. The safe use of surface waters for drinking water production and recreation necessitates assessment of toxigenic cyanobacteria. We have developed simple and reliable sample preparation and qPCR methods to detect microcystin-producing strains of three major bloom-forming genera, Anabaena, Microcystis and Planktothrix. The mcyB second thiolation motif, previously not recognized as a potential target for qPCR, was used as a basis for primer and genus-specific probe design. Assay specificity and sensitivity was confirmed with cultured cyanobacterial strains and the effect of different sample preparation methods on quantification was investigated. Sample filtration and cell lysis reduced assay time and resulted in more efficient amplification compared to DNA extraction. Positive correlation (p<0.005) between mcyB copy numbers and microcystin concentrations was observed in environmental samples. The results encourage the use of qPCR in water risk management.
Subject(s)
Environmental Monitoring/methods , Real-Time Polymerase Chain Reaction , Anabaena/classification , Anabaena/genetics , Animals , Cyanobacteria/classification , Cyanobacteria/genetics , DNA, Bacterial/chemistry , Microcystins/metabolism , Microcystis/genetics , Risk Management/methodsABSTRACT
The extract of UIC 10035, a strain obtained from a sample collected near the town of Homestead, South Florida, showed antiproliferative activity against MDA-MB-435 cells. Bioassay-guided fractionation led to the isolation of a series of cyclic lipodecapeptides, named minutissamides E-L (1-8). The planar structures were determined by analysis of HRESIMS, tandem MS, and 1D and 2D NMR data, and the stereoconfigurations were assigned by LC-MS analysis of the Marfey's derivatives after acid hydrolysis. Minutissamides E-L (1-8) exhibited antiproliferative activity against MDA-MB-435 cells with IC(50) values ranging between 1 and 10 µM. The structures of minutissamides E-L (1-8) were closely related with those of the previously reported lipopeptides, puwainaphycins A-E and minutissamides A-D, characterized by the presence of a lipophilic ß-amino acid and three non-standard amino acids NMeAsn, OMeThr and Dhb (α,ß-dehydro-α-aminobutyric acid). The strain UIC 10035 was designated as cf. Anabaena sp. on the basis of morphological and 16S rRNA gene sequence analyses.
Subject(s)
Anabaena/chemistry , Lipopeptides/chemistry , Anabaena/classification , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Fresh Water/microbiology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Lipopeptides/isolation & purification , Lipopeptides/toxicity , Magnetic Resonance Spectroscopy , Molecular Conformation , Phylogeny , RNA, Ribosomal, 16S/genetics , Tandem Mass SpectrometryABSTRACT
DNA-binding proteins from starved cells (Dps), which are encoded by many bacterial genomes, protect genomic DNA via non-specific DNA binding, as well as inhibition of free radical formation by chelating Fe(II). In the filamentous cyanobacterium Anabaena, the second gene (lti46.2) in the low temperature-induced gene operon lti46 in strain M3 was found to encode a homologue of Dps, but for a long time this gene remained poorly characterized. A gene cluster, all0459-all0458-all0457, was found later to be 100% identical to the lti46 gene cluster in a closely related strain, PCC 7120. In the present study, we detected ferroxidase activity of the Lti46.2/All0458 protein, which formed a dodecamer, as found in other Dps proteins. In addition, three homologues of all0458 were found in strain PCC 7120, namely, all1173, alr3808 and all4145. We analysed expression of the lti46 or all0459-8-7 gene cluster in both strains, M3 and PCC 7120, and confirmed its induction by low temperature. We found that the All0458-GFP fusion protein and the All1173-GFP fusion protein were localized to the nucleoids. In the all0458 null mutant, the transcript of the alr3808 gene accumulated. These results suggest that there might be complex cooperation of various members of the dps family in protecting the genome from environmental stresses such as changing temperature.
Subject(s)
Anabaena variabilis/genetics , Anabaena/genetics , Bacterial Proteins/metabolism , Cold Temperature , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial , Anabaena/classification , Anabaena/metabolism , Anabaena/physiology , Anabaena variabilis/metabolism , Anabaena variabilis/physiology , Bacterial Proteins/genetics , Ceruloplasmin/metabolism , DNA-Binding Proteins/genetics , Heat-Shock Response , MutationABSTRACT
The main goal of this study was to determine the distribution of potentially toxic cyanobacteria in 39 selected Polish water bodies. From the water bodies with blooms and also from those in which blooms were not visible 87 samples were investigated. For the first time samples from ponds localized in villages with high agricultural activities were included. Lakes for which microcystin concentrations had been determined before were included as a reference for the research. The detection of cyanobacteria was conducted by microscopic observation as well as by PCR amplification of the rpoC1 gene fragment. Cyanobacteria were present in 75 out of 87 samples. The presence of potentially toxic cyanobacteria was detected by amplification of the mcyB and mcyE genes, which are involved in the biosynthesis of microcystins. Both genes were detected in 7 out of 9 blooms investigated. In the case of samples collected from water bodies in which blooms were not observed, the mcyB and mcyE genes were detected in 20 out of 36. In order to identify the cyanobacteria occurring in selected reservoirs, 16S plus ITS clone libraries were constructed. The method allowed distinguishing 18 different genotypes. After sequence analysis, cyanobacteria belonging to genera Microcystis, Planktothrix, Anabaena, Pseudanabaena, Synechocystis, Synechococcus and Woronichinia were identified. Results confirmed the usefulness of the rpoC1 and mcy genes for monitoring water bodies and detection of potentially toxic cyanobacteria. Application of molecular markers allowed detecting potentially toxic cyanobacteria before the bloom was visible. This is the first comprehensive study concerning cyanobacteria present in different types of Polish water bodies performed using molecular markers.
Subject(s)
Cyanobacteria/classification , Cyanobacteria/isolation & purification , Fresh Water/microbiology , Anabaena/classification , Anabaena/genetics , Anabaena/isolation & purification , Bacterial Proteins/genetics , Cyanobacteria/genetics , Environmental Monitoring/methods , Microcystins/analysis , Microcystins/genetics , Microcystis/classification , Microcystis/genetics , Microcystis/isolation & purification , Poland , Polymerase Chain Reaction , Synechococcus/classification , Synechococcus/genetics , Synechococcus/isolation & purification , Synechocystis/classification , Synechocystis/genetics , Synechocystis/isolation & purificationABSTRACT
Recent studies indicate genetic diversity of cyanobacteria in eutrophic lakes is not represented well by culture collections or morphology. Yet, few studies have investigated genetic richness and evenness of cyanobacteria using culture-independent methods. We compared the genetic structure of cyanobacteria supported by four neighboring eutrophic lakes during the ice-free season. The partial phycobilincpcB/A genes plus intergenic spacer (PC-IGS) was used as a genetic marker.Sequences were phylogeneticallygrouped by maximum likelihood into genotypes representing sub-genera of the major taxa. Genotypes fell into genera commonly observed by microscopy in these lakes including Microcystis, Aphanizomenon, Chroococcus, Anabaena, and Cylindrospermopsis. Only three genotypes were shared among all four lakes, despite significant water flowage between lakes.A Parsimony P-test indicated lakes were significantly (p=0.01) clustered on the maximum likelihood tree. Pairwise differences using Unifrac distance were moderately or not significant. Analysis of molecular variance (AMOVA) indicated genetic variation among all genotypes (φ=0.06, p<0.001) and 94% of variability occurred within lakes rather than between lakes (6%), explaining the lack of pairwise differences between lakes. Lorenze curves of genotype abundance in each lake showed genetic structure was only moderately uneven (Gini coefficients of 0.37-0.5) indicating lakes did not support dominant genotypes. Overall, results from this study suggest diversity of cyanobacteria is shaped by heterogeneity within lakes (temporally or spatially) and relatively even population structures.
Subject(s)
Cyanobacteria/classification , Lakes/microbiology , Anabaena/classification , Anabaena/genetics , Anabaena/growth & development , Base Sequence , Cyanobacteria/genetics , Cyanobacteria/growth & development , Eutrophication , Genetic Variation , Genotype , Microcystis/classification , Microcystis/genetics , Microcystis/growth & development , Molecular Sequence DataABSTRACT
Cyanobacterial mass occurrences are widespread and often contain hepatotoxic, i.e. microcystin- and nodularin-producing, species. Nowadays, detection of microcystin (mcy) and nodularin synthetase (nda) genes is widely used for the recognition of toxic cyanobacterial strains in environmental water samples. Chip assay presented here combines ligation detection reaction and hybridization on a universal microarray to detect and identify the mcyE/ndaF genes of five cyanobacterial genera specifically and sensitively. Thus, one chip assay can reveal the co-occurrence of several hepatotoxin producers. The presented quantitative real-time PCR method is used for the detection of either microcystin-producing Anabaena or Microcystis. Determination of the mcyE-gene copy numbers allows the identification of the dominant producer genus in the sample.
Subject(s)
Anabaena/isolation & purification , Bacterial Proteins/analysis , Microarray Analysis/methods , Microcystins/analysis , Microcystis/isolation & purification , Polymerase Chain Reaction/methods , Anabaena/classification , Anabaena/genetics , Bacterial Proteins/genetics , Bacterial Typing Techniques , DNA Primers/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Gene Dosage , Genes, Bacterial , Microcystis/classification , Microcystis/genetics , Peptides, Cyclic/analysis , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/geneticsABSTRACT
Heterocysts are specialized cells required for aerobic fixation of dinitrogen by certain filamentous cyanobacteria. Numerous genes involved in the differentiation and function of heterocysts in Anabaena sp. strain PCC 7120 have been identified by mutagenizing and screening for mutants that require fixed nitrogen for growth in the presence of oxygen. We have verified that 10 Anabaena sp. genes, all1338, all1591, alr1728, all3278, all3520, all3582, all3850, all4019, alr4311, and all4388, identified initially by transposon mutagenesis, are such genes by complementing or reconstructing the original mutation and by determining whether the mutant phenotype might be due to a polar effect of the transposon. Elucidation of the roles of these genes should enhance understanding of heterocyst biology.
Subject(s)
Anabaena/growth & development , Anabaena/metabolism , Bacterial Proteins/genetics , Nitrogen Fixation , Aerobiosis , Anabaena/classification , Anabaena/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Nitrogen/metabolism , PhylogenyABSTRACT
The PipX factor is a regulatory protein that seems to occur only in cyanobacteria. In the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120, open reading frame (ORF) asr0485, identified as the pipX gene, is expressed mainly under conditions of combined-nitrogen deprivation dependent on the global N regulator NtcA and the heterocyst-specific regulator HetR. Primer extension and 5' rapid amplification of cDNA ends (RACE) analyses detected three transcription start points corresponding to a canonical NtcA-activated promoter (to which direct binding of NtcA was observed), an NtcA- and HetR-dependent promoter, and a consensus-type promoter, the last with putative -35 and -10 determinants. Activation of pipX took place in cells differentiating into heterocysts at intermediate to late stages of the process. Accordingly, disruption of pipX led to impaired diazotrophic growth, reduced nitrogenase activity, and impaired activation of the nitrogenase structural genes. The nitrogenase activity of the mutant was low under oxic conditions, likely resulting from inefficient protection against oxygen. In line with this, the activation of the coxB2A2C2 and coxB3A3C3 operons, encoding heterocyst-specific terminal respiratory oxidases responsible for internal oxygen removal, was deficient in the pipX mutant. Therefore, the Anabaena PipX factor shows a spatiotemporal specificity contributing to normal heterocyst function, including full activation of the nitrogenase structural genes and genes of the nitrogenase-protective features of the heterocyst.