Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Genomic signatures of Lake Erie bacteria suggest interaction in the Microcystis phycosphere.

Microbial interactions in harmful algal bloom (HAB) communities have been examined in marine systems, but are poorly studied in fresh waters. To investigate HAB-microbe interactions, we isolated bacteria with close associations to bloom-forming cyanobacteria, Microcystis spp., during a 2017 bloom in the western basin of Lake Erie. The genomes of five isolates (Exiguobacterium sp. JMULE1, Enterobacter sp. JMULE2, Deinococcus sp. JMULE3, Paenibacillus sp. JMULE4, and Acidovorax sp. JMULE5.) were sequenced on a PacBio Sequel system. These genomes ranged in size from 3.1 Mbp (Exiguobacterium sp. JMULE1) to 5.7 Mbp (Enterobacter sp. JMULE2). The genomes were analyzed for genes relating to critical metabolic functions, including nitrogen reduction and carbon utilization. All five of the sequenced genomes contained genes that could be used in potential signaling and nutrient exchange between the bacteria and cyanobacteria such as Microcystis. Gene expression signatures of algal-derived carbon utilization for two isolates were identified in Microcystis blooms in Lake Erie and Lake Tai (Taihu) at low levels, suggesting these organisms are active and may have a functional role during Microcystis blooms in aggregates, but were largely missing from whole water samples. These findings build on the growing evidence that the bacterial microbiome associated with bloom-forming algae have the functional potential to contribute to nutrient exchange within bloom communities and interact with important bloom formers like Microcystis.

Isolation of axenic cyanobacterium and the promoting effect of associated bacterium on axenic cyanobacterium.

BACKGROUND: Harmful cyanobacterial blooms have attracted wide attention all over the world as they cause water quality deterioration and ecosystem health issues. Microcystis aeruginosa associated with a large number of bacteria is one of the most common and widespread bloom-forming cyanobacteria that secret toxins. These associated bacteria are considered to benefit from organic substrates released by the cyanobacterium. In order to avoid the influence of associated heterotrophic bacteria on the target cyanobacteria for physiological and molecular studies, it is urgent to obtain an axenic M. aeruginosa culture and further investigate the specific interaction between the heterotroph and the cyanobacterium. RESULTS: A traditional and reliable method based on solid-liquid alternate cultivation was carried out to purify the xenic cyanobacterium M. aeruginosa FACHB-905. On the basis of 16S rDNA gene sequences, two associated bacteria named strain B905-1 and strain B905-2, were identified as Pannonibacter sp. and Chryseobacterium sp. with a 99 and 97% similarity value, respectively. The axenic M. aeruginosa FACHB-905A (Microcystis 905A) was not able to form colonies on BG11 agar medium without the addition of strain B905-1, while it grew well in BG11 liquid medium. Although the presence of B905-1 was not indispensable for the growth of Microcystis 905A, B905-1 had a positive effect on promoting the growth of Microcystis 905A. CONCLUSIONS: The associated bacteria were eliminated by solid-liquid alternate cultivation method and the axenic Microcystis 905A was successfully purified. The associated bacterium B905-1 has the potentiality to promote the growth of Microcystis 905A. Moreover, the purification technique for cyanobacteria described in this study is potentially applicable to a wider range of unicellular cyanobacteria.

Cyanobacterial Classification with the Toxicity Using MALDI Biotyper.

An abnormal growth of cyanobacteria in eutrophicated freshwaters can cause various environmental problems. In particular, Microcystis producing hepatotoxic cyclic heptapeptides microcystins (MCs) has been globally observed. Recent studies have demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a rapid classification of cyanobacteria; however, they have not fully considered the toxicity yet. In this study, we have performed MALDI-TOF MS for intact cyanobacterial cells using Biotyper software and optimized their conditions to achieve cyanobacterial classification with the toxicity. The detection mass range used for Biotyper was extended to cover small molecules, but their intense ions were suppressed as a function of the used instrument Autoflex Speed, which enabled simultaneous observations of large molecular fingerprints and small MCs with comparable ion intensity. Hierarchical clustering of mass spectra obtained under the optimized conditions differentiated toxic and non-toxic clusters of Microcystis strains and furthermore formed a tight cluster of non-toxic strains possessing the MC biosynthesis gene mcyG. Spectral libraries were expanded to >30 genera (>80 strains) under the default and optimized conditions to improve the confidence of cyanobacterial classification. Consequently, spectral library searching allowed for characterization of cyanobacteria from a field sample as mixed toxic and non-toxic Microcystis cells, without isolating those cells.

Cylindrospermopsin- and Deoxycylindrospermopsin-Producing Raphidiopsis raciborskii and Microcystin-Producing Microcystis spp. in Meiktila Lake, Myanmar.

Meiktila Lake is a shallow reservoir located close to Meiktila city in central Myanmar. Its water is used for irrigation, domestic purposes and drinking water. No detailed study of the presence of cyanobacteria and their potential toxin production has been conducted so far. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Meiktila Lake, water samples were collected in March and November 2017 and investigated for physico-chemical and biological parameters. Phytoplankton composition and biomass determination revealed that most of the samples were dominated by the cyanobacterium Raphidiopsis raciborskii. In a polyphasic approach, seven isolated cyanobacterial strains were classified morphologically and phylogenetically as R. raciborskii, and Microcystis spp. and tested for microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins and anatoxins by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). ELISA and LC-MS analyses confirmed CYNs in three of the five Raphidiopsis strains between 1.8 and 9.8 µg mg-1 fresh weight. Both Microcystis strains produced MCs, one strain 52 congeners and the other strain 20 congeners, including 22 previously unreported variants. Due to the presence of CYN- and MC-producing cyanobacteria, harmful effects on humans, domestic and wild animals cannot be excluded in Meiktila Lake.

Comparative genomics analysis of c-di-GMP metabolism and regulation in Microcystis aeruginosa.

BACKGROUND: Cyanobacteria are of special concern because they proliferate in eutrophic water bodies worldwide and affect water quality. As an ancient photosynthetic microorganism, cyanobacteria can survive in ecologically diverse habitats because of their capacity to rapidly respond to environmental changes through a web of complex signaling networks, including using second messengers to regulate physiology or metabolism. A ubiquitous second messenger, bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has been found to regulate essential behaviors in a few cyanobacteria but not Microcystis, which are the most dominant species in cyanobacterial blooms. In this study, comparative genomics analysis was performed to explore the genomic basis of c-di-GMP signaling in Microcystis aeruginosa. RESULTS: Proteins involved in c-di-GMP metabolism and regulation, such as diguanylate cyclases, phosphodiesterases, and PilZ-containing proteins, were encoded in M. aeruginosa genomes. However, the number of identified protein domains involved in c-di-GMP signaling was not proportional to the size of M. aeruginosa genomes (4.97 Mb in average). Pan-genome analysis showed that genes involved in c-di-GMP metabolism and regulation are conservative in M. aeruginosa strains. Phylogenetic analysis showed good congruence between the two types of phylogenetic trees based on 31 highly conserved protein-coding genes and sensor domain-coding genes. Propensity for gene loss analysis revealed that most of genes involved in c-di-GMP signaling are stable in M. aeruginosa strains. Moreover, bioinformatics and structure analysis of c-di-GMP signal-related GGDEF and EAL domains revealed that they all possess essential conserved amino acid residues that bind the substrate. In addition, it was also found that all selected M. aeruginosa genomes encode PilZ domain containing proteins. CONCLUSIONS: Comparative genomics analysis of c-di-GMP metabolism and regulation in M. aeruginosa strains helped elucidating the genetic basis of c-di-GMP signaling pathways in M. aeruginosa. Knowledge of c-di-GMP metabolism and relevant signal regulatory processes in cyanobacteria can enhance our understanding of their adaptability to various environments and bloom-forming mechanism.

Distribution of the Harmful Bloom-Forming Cyanobacterium, Microcystis aeruginosa, in 88 Freshwater Environments across Japan.

Microcystis aeruginosa was quantitatively surveyed in 88 freshwater environments across Japan within 3| |weeks in 2011. In order to clarify the distribution pattern of M. aeruginosa at the intra-species level, three major genotypes, which were defined by 16S-23S rRNA inter-transcribed-spacer (ITS) regions, were selectively detected using quantitative real-time PCR assays. Of the 68 sites at which the Microcystis intergenic-spacer region of the phycocyanin (IGS-PC) gene was detected, the M. aeruginosa morphotype-related genotype (MG1) dominated in 41 sites, followed by the non-toxic M. wesenbergii-related genotype (MG3). A correlation analysis showed that total nitrogen and phosphate positively correlated with the abundance of IGS-PC, which positively correlated with microcystin synthetase gene abundance. A redundancy analysis of genotype compositions showed that pH positively correlated with the dominance of MG3 and negatively correlated with MG1, i.e., both toxic and non-toxic genotypes. Our survey of Microcystis populations over a wide area revealed that MG1 is a dominant genotype in Japan.

Identification and characterization of the dominant Microcystis sp. cyanobacteria detected in Lake Dong Ting, China.

The presence of cyanobacteria in drinking water, aquatic foods and bathing water has created a significant major problem to global public health as these toxins induce damage in various organ including liver, cardiovascular, intestinal and central nervous systems. Although the morphologic, phylogenetic and toxicogenetic characteristics of cyanobacteria were identified in several lakes in China, many freshwater sources such as Dong Ting Lake, Hunan Province, China remain to be determined. Since the presence of these cyanobacteria may potentially affect human health, the aim of this study was to isolate, identify and characterize the most frequent occurring bloom-forming cyanobacteria in Dong Ting Lake, Hunan Province, China, which can provide information on the safety of utilization of this water source for drinking water, agriculture and recreation. Samples collected from the surface water of Dong Ting Lake were subjected to serial dilution in the lab for morphological analysis. Data demonstrated the morphological features were 2-5 µm diameters with rounded shapes and green color resembling Microcystis sp. The isolated cyanobacterial strain obtained from surface water samples in Dong Ting Lake was termed Microcystis sp. YFM2. The MC concentration was detected by enzyme-linked immunosorbent assay (ELISA) and found to be 92.88 µg/107 cells in Microcystis sp. YFM2. By polymerase chain reaction (PCR) results indicated that Microcystis sp. YFM2 isolated from Dong Ting Lake contained synthetase genes (mcyA-C). Our findings indicated that the dominant cyanobacteria Microcystis sp. YFM2 isolated from the freshwater Dong Ting Lake demonstrated morphologic, phylogenetic and toxicogenetic properties resembling a toxin generating cyanobacterium. Based upon this knowledge, it is essential to monitor the use of this Lake for future domestic, agricultural and recreational purposes.

Obtaining Genome Sequences of Mutualistic Bacteria in Single Microcystis Colonies.

Cells of Microcystis are associated with heterotrophic bacteria and organized in colonies in natural environment, which are basic elements in the mass occurrence of cyanobacterial species. Analyzing these colonies by using metagenomics is helpful to understand species composition and relationship. Meanwhile, the difference in population abundance among Microcystis colonies could be used to recover genome bins from metagenome assemblies. Herein, we designed a pipeline to obtain high-quality genomes of mutualistic bacteria from single natural Microcystis colonies. Single colonies were lysed, and then amplified by using multiple displacement amplification to overcome the DNA quantity limit. A two-step assembly was performed after sequencing and scaffolds were grouped into putative bins based on their differential-coverage among species. We analyzed six natural colonies of three prevailing Microcystis species from Lake Taihu. Clustering results proved that colonies of the same species were similar in the microbial community composition. Eight putative population genome bins with wide bacterial diversity and different GC content were identified based on coverage difference among colonies. At the phylum level, proteobacteria was the most abundant besides cyanobacteria. Six of the population bins were further refined into nearly complete genomes (completeness > 90%).

Coherence of Microcystis species revealed through population genomics.

Microcystis is a genus of freshwater cyanobacteria, which causes harmful blooms in ecosystems worldwide. Some Microcystis strains produce harmful toxins such as microcystin, impacting drinking water quality. Microcystis colony morphology, rather than genetic similarity, is often used to classify Microcystis into morphospecies. Yet colony morphology is a plastic trait, which can change depending on environmental and laboratory culture conditions, and is thus an inadequate criterion for species delineation. Furthermore, Microcystis populations are thought to disperse globally and constitute a homogeneous gene pool. However, this assertion is based on relatively incomplete characterization of Microcystis genomic diversity. To better understand these issues, we performed a population genomic analysis of 33 newly sequenced genomes mainly from Canada and Brazil. We identified 17 Microcystis clusters of genomic similarity, five of which correspond to monophyletic clades containing at least three newly sequenced genomes. Four out of these five clades match to named morphospecies. Notably, M. aeruginosa is paraphyletic, distributed across 12 genomic clusters, suggesting it is not a coherent species. A few clades of closely related isolates are specific to a unique geographic location, suggesting biogeographic structure over relatively short evolutionary time scales. Higher homologous recombination rates within than between clades further suggest that monophyletic groups might adhere to a Biological Species-like concept, in which barriers to gene flow maintain species distinctness. However, certain genes-including some involved in microcystin and micropeptin biosynthesis-are recombined between monophyletic groups in the same geographic location, suggesting local adaptation.

Isolation, molecular identification, and characterization of a unique toxic cyanobacterium Microcystis sp. found in Hunan Province, China.

Global proliferation of cyanobacterial blooms associated with climate change and eutrophication constitutes a serious environmental threat. In Hunan Province a freshwater pond located in Changsha City was found to contain high concentrations of cyanobacteria, however, the characteristics of these cyanobacteria at present are not known. This study thus aimed to isolate, identify the most common bloom-forming cyanobacteria in this region and determine the toxigenic characteristics of the predominant cyanobacteria. The cyanobacteria were isolated by serial dilution and identified using polymerase chain reaction (PCR). The cyanotoxins generated by the cyanobacterium were detected using high-performance liquid chromatography with an ultra-high resolution LTQ Orbitrap Velos Pro ETD mass spectrometry equipped with electrospray ionization interface (HPLC-ESI-MS). One  species of cyanobacterium was isolated and identified as Microcystis sp. YFM1 according to the sequence of the 16S ribosome deoxyribonucleic acid (16S rDNA). It was found that this cyanobacterium contained microcystin synthetase B gene (mcyB) and produced three types of cyanotoxins including microcystin-LR, RR and YR. Our findings indicate that the Microcystis sp. YFM1 isolated from the freshwater pond in Hunan Province exhibits unique characteristics distinguishable from other known cyanobacteria.

Variability in a permanent cyanobacterial bloom: species-specific responses to environmental drivers.

Cyanobacterial blooms are characterized by intense growth of one or few species that will dominate the phytoplankton community for periods of few months to an entire year or more. However, even during persistent blooms, important seasonal changes among dominant species can be observed. Pampulha reservoir is a tropical eutrophic reservoir presenting permanent blooms. To identify the main species in this environment, a closer analysis performed by microscopy and 16S-rRNA DGGE revealed Cylindrospermopsis raciborskii as highly dominant throughout the year. The second most abundant group comprised species belonging to the Microcystis genus. They followed a well-defined seasonal pattern described by interesting species-specific ecological trends. During thermal stratification in the rainy/warmer season, C. raciborskii dominated in the water column, while Microcystis spp. were abundant at the end of the dry season, a period characterized by higher total phosphorus concentrations. Phylogenetic analyses confirmed the two dominant taxa and their seasonal trends. The results showed that cyanobacteria major controlling factors were strongly species dependent, shifting from physical/climate related (stratification) to more chemical driven (nutrients/eutrophication). Identifying these drivers is therefore essential for the understanding of the bloom dynamics and the real risks associated with each species, and to eventually adopt the most appropriate and effective management strategies.

Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake.

The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejér county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6 weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.

Distinct Bloom Dynamics of Toxic and Non-toxic Microcystis (Cyanobacteria) Subpopulations in Hoedong Reservoir (Korea).

Despite the importance of understanding the bloom mechanisms that influence cyanobacterial toxin production, the dynamics of toxic Microcystis subpopulations are largely unknown. Here, we quantified both toxic and entire (i.e., toxic and non-toxic) Microcystis populations based on the microcystin synthetase E (mcyE) and 16S ribosomal RNA genes. Samples were collected from pelagic water and sediments twice per week from October to December 2011, and we investigated the effects of physicochemical factors (pH, water temperature, dissolved oxygen, nutrients, etc.) and biological factors (ciliates and zooplankton) on the abundance of toxic and non-toxic Microcystis. During the study period, Microcystis blooms were composed of toxic and non-toxic subpopulations. Resting stage Microcystis in sediment may be closely linked to Microcystis populations in pelagic water and may contribute to the toxic subpopulation composition in surface Microcystis blooms. In pelagic water, the toxic and entire Microcystis population had a significant positive correlation with the pH and water temperature (p < 0.05). However, their responses to changes in environmental factors were thought to be distinct. The ratio of the toxic to non-toxic Microcystis subpopulations was significantly (p < 0.05) enhanced by a lower pH and water temperature and an increase in protozoan grazers, reflecting environmental stresses. These results suggest that the toxic and non-toxic subpopulations of Microcystis have distinct tolerance levels against these stressors. The intracellular microcystin (MC) concentration was positively associated with the abundance of the mcyE-positive Microcystis. By comparison, the MC concentration in pelagic water body (extracellular) increased when Microcystis was lysed due to environmental stresses.

Metatranscriptomics analysis of cyanobacterial aggregates during cyanobacterial bloom period in Lake Taihu, China.

Molecular mechanism of interaction between the bloom-forming cyanobacterial species and attached microbios within cyanobacterial aggregates has not been elucidated yet and understanding of which would help to unravel the cyanobacteria bloom-forming mechanism. In this study, we profiled the metabolically active community by high-throughput metatranscriptome sequencing from cyanobacterial aggregates during cyanobacterial bloom period in Lake Taihu, China. A total of 308 million sequences were obtained using the HiSeq 2500 sequencing platform, which provided a great sequence coverage to carry out the in-depth taxonomic classification, functional classification, and metabolic pathway analysis of the cyanobacterial aggregates. The results show that bacteria dominated in cyanobacterial aggregates, accounting for more than 96.66% of total sequences. Microcystis was the most abundant genus, accounted for 26.80% of total assigned sequences at the genus level in cyanobacterial aggregates community; however, Proteobacteria (46.20%) was found to be as the most abundant active bacterial populations at the phylum level. More importantly, nitrogen, phosphonate, and phosphinate metabolism which associated with eutrophication were found in this study. Especially, the enzymes and organisms relating to denitrification and anammox of nitrogen metabolism, which reduced nitrogen concentration by reducing nitrate to nitrogen to inhibit the eutrophication, were first discovered in Lake Taihu during cyanobacterial bloom period. The present study provides a snapshot of metatranscriptome for cyanobacterial aggregates in Lake Taihu and demonstrates that cyanobacterial aggregates could play a key role in the nitrogen cycle in eutrophic water.

Community composition specificity and potential role of phosphorus solubilizing bacteria attached on the different bloom-forming cyanobacteria.

The abundance, phosphorus solubilizing ability and community composition of phosphorus solubilizing bacteria (PSB) attached on two bloom-forming cyanobacteria, Microcystis and Anabeana, were investigated in Guanqiao ponds in 2014 and Lake Chaohu in 2015 and 2016. Thirty organic phosphate-mineralizing bacteria (OPB) and eighteen inorganic phosphate-solubilizing bacteria (IPB) isolated from Guanqiao ponds and Lake Chaohu were identified. The community compositions of PSB attached on Microcystis and Anabeana were found to be entirely different. Some PSB were found to be shared by OPB and IPB, especially the species attached on Microcystis, such as Rhizobium sp. Compared to the PSB attached on Anabeana, the PSB attached on Microcystis showed the lower numbers, higher phosphorus solubilizing ability and extensive substrate adaptability. This indicated that the PSB were important for the growth of Microcystis through meeting soluble reactive phosphorus (SRP) demand, which was further supported by the data from Guanqiao ponds where succession process from Anabeana to Microcystis was recorded. All these facts can explain why the succession from Anabeana to Microcystis frequently occurred in shallow eutrophic lakes. Therefore, the attached PSB should be considered as a crucial contributor on algal growth, succession and collapse, depending on algal species.

Predicting blooms of toxic cyanobacteria in eutrophic lakes with diverse cyanobacterial communities.

We investigated possibility of predicting whether blooms, if they occur, would be formed of microcystin-producing cyanobacteria. DGGE analysis of 16S-ITS and mcyA genes revealed that only Planktothrix and Microcystis possessed mcy-genes and Planktothrix was the main microcystin producer. qPCR analysis revealed that the proportion of cells with mcy-genes in Planktothrix populations was almost 100%. Microcystin concentration correlated with the number of potentially toxic and total Planktothrix cells and the proportion of Planktothrix within all cyanobacteria, but not with the proportion of cells with mcy-genes in total Planktothrix. The share of Microcystis cells with mcy-genes was low and variable in time. Neither the number of mcy-possessing cells, nor the proportion of these cells in total Microcystis, correlated with the concentration of microcystins. This suggests that it is possible to predict whether the bloom in the Masurian Lakes will be toxic based on Planktothrix occurrence. Two species of toxin producing Planktothrix, P. agardhii and P. rubescens, were identified by phylogenetic analysis of 16S-ITS. Based on morphological and ecological features, the toxic Planktothrix was identified as P. agardhii. However, the very high proportion of cells with mcy-genes suggests P. rubescens. Our study reveals the need of universal primers for mcyA genes from environment.

The molecular ecology of Microcystis sp. blooms in the San Francisco Estuary.

Harmful blooms of the cyanobacterium Microcystis sp. have become increasingly pervasive in the San Francisco Estuary Delta (USA) since the early 2000s and their rise has coincided with substantial decreases in several important fish species. Direct and indirect effects Microcystis blooms may have on the Delta food web were investigated. The Microcystis population was tracked for 2 years at six sites throughout the Delta using quantitative PCR. High-throughput amplicon sequencing and colony PCR sequencing revealed the presence of 10 different strains of Microcystis, including 6 different microcystin-producing strains. Shotgun metagenomic analysis identified a variety of Microcystis secondary metabolite pathways, including those for the biosynthesis of: aeruginosin, cyanopeptolin, microginin, microviridin and piricyclamide. A sizable reduction was observed in microbial community diversity during a large Microcystis bloom (H' = 0.61) relative to periods preceding (H' = 2.32) or following (H' = 3.71) the bloom. Physicochemical conditions of the water column were stable throughout the bloom period. The elevated abundance of a cyanomyophage with high similarity to previously sequenced isolates known to infect Microcystis sp. was implicated in the bloom's collapse. Network analysis was employed to elucidate synergistic and antagonistic relationships between Microcystis and other bacteria and indicated that only very few taxa were positively correlated with Microcystis.

Acidovorax lacteus sp. nov., isolated from a culture of a bloom-forming cyanobacterium (Microcystis sp.).

A novel Gram-negative, rod-shaped and motile bacterial strain, designated strain M36T, was isolated from a culture of a bloom-forming cyanobacterium, Microcystis sp., collected from a eutrophic lake in Korea. Its taxonomic position was investigated by using a polyphasic taxonomic approach. The isolate was found to grow aerobically at 15-42 °C (optimum 25 °C), pH 7.0-11.0 (optimum pH 8.0) and in the presence of 0-1.0% (w/v) NaCl (optimum 0% NaCl) on R2A medium. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain M36T is closely related to Acidovorax anthurii DSM 16745T (98.1%), Acidovorax konjaci DSM 7481T (97.7%) and Acidovorax avenae DSM 7227T (97.0%) and also formed a clear phylogenetic lineage with other Acidovorax species. DNA-DNA relatedness between strain M36T and the closely related species of the genus Acidovorax was <30%. The major fatty acid components identified included summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:0 ω7c and/or C18:0 ω6c). The DNA G+C content of strain M36T was determined to be 66.8 mol%. Based on above polyphasic evidence, strain M36T is concluded to represent a new species of genus Acidovorax, for which the name Acidovorax lacteus sp. nov. is proposed. The type strain is M36T (=KCTC 52220T = JCM 31890T).

Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes.

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics.