High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Phenotypic and Genotypic Comparison of Symbiotic and Free-Living Cyanobacteria from a Single Field Site

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.     

High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.     

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat.

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.     

应用RT-qPCR技术定量检测湖泊水体中蓝藻方法的比较

利用RT-qPCR技术建立了对湖泊水体中的微囊藻和蓝藻的SYBR Green Ⅰ荧光定量PCR检测方法,在所建立的方法中,对以微囊藻藻蓝蛋白基因、蓝藻16S rRNA基因、微囊藻16S rRNA基因分别作为RT-qPCR检测的目的基因所得结果进行了比较,并对实验室培养的微囊藻和太湖的环境样品进行了检测。结果表明,用藻蓝蛋白基因作为检测目的基因,以M.aeruginosa PCC 7806基因组DNA作为标准品的测定方法与显微镜计数的结果有较好的相关性和一致性,并具有简便、快速、特异性高的特点,可以满足检测的要求。     

Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the generaLeptolyngbya andOscillatoria were cloned and sequenced. For the genusLeptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strainsLeptolyngbya foveolarum Komárek 1964/112,Leptolyngbya sp. VRUC 135 Albertano 1985/1, andLeptolyngbya boryanum PCC 73110 belong to the same cluster. StrainOscillatoria cf.corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria.Oscillatoria agardhii CYA 18, which is a representative of planktonicOscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.     

An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.      

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Gene copy number variation and its significance in cyanobacterial phylogeny

ABSTRACT: BACKGROUND: In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. RESULTS: We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic istances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions: A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria.     

Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status

In lakes, benthic micro-algae and cyanobacteria (periphyton) can contribute significantly to total primary productivity and provide important food sources for benthic invertebrates. Despite recognition of their importance, few studies have explored the diversity of the algal and cyanobacterial composition of periphyton mats in temperate lakes. In this study, we sampled periphyton from three New Zealand lakes: Tikitapu (oligotrophic), ōkāreka (mesotrophic) and Rotoiti (eutrophic). Statistical analysis of morphological data showed a clear delineation in community structure among lakes and highlighted the importance of cyanobacteria. Automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity. Despite the close geographic proximity of the lakes, cyanobacterial species differed markedly. The 16S rRNA gene sequence analysis identified eight cyanobacterial OTUs. A comparison with other known cyanobacterial sequences in GenBank showed relatively low similarities (91-97%). Cyanotoxin analysis identified nodularin in all mats from Lake Tikitapu. ndaF gene sequences from these samples had very low (≤ 89%) homology to sequences in other known nodularin producers. To our knowledge, this is the first detection of nodularin in a freshwater environment in the absence of Nodularia. Six cyanobacteria species were isolated from Lake Tikitapu mats. None were found to produce nodularin. Five of the species shared low (< 97%) 16S rRNA gene sequence similarities with other cultured cyanobacteria.     

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans.

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.     

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans.

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.     

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.     

16S rRNA-Targeted identification of cyanobacterial genera using oligonucleotide-probes immobilized on bacterial magnetic particles

16S rRNA-targeted identification of cyanobacterial genera, Anabaena,Microcystis, Nostoc, Oscillatoria, Synechococcus wasdeveloped using bacterial magnetic particles (BMPs). 16S rRNA-targetedcapture probes designed from the genus specific region of the 16S rRNAsequence were immobilized on BMPs. Identification of cyanobacteria wasperformed by a sandwich hybridization using the capture probes – BMPconjugates and a digoxigenin (DIG)-labeled detector probe complementaryto the highly conserved 16S rRNA sequence for cyanobacteria. Theluminescence intensity of the probe/target-BMP hybrids was measured afterreaction with alkaline phosphatase conjugated anti-DIG antibody. Fivespecies of cyanobacteria from five different genera were successfullydiscriminated using this magnetic capture system.     

Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

Testing of primers for the study of cyanobacterial molecular diversity by DGGE

Denaturing Gradient Gel electrophoresis (DGGE) is a PCR-based technique which is widely used in the study of microbial communities. Here, the use of the three specific 16S rRNA cyanobacterial specific primers CYA359F, CYA781R(a) and CYA781R(b) on the assessment of the molecular diversity of cyanobacterial communities is examined. Assignments of the reverse primers CYA781R(a) and CYA781R(b) with cyanobacterial strain sequences showed that the former preferentially targets filamentous cyanobacteria whereas the latter targets unicellular cyanobacteria. The influence of the GC clamp position on the forward or on reverse primer and the use of the two reverse primers separately or in equimolar mixture were investigated. Three environmental samples were subjected to amplification with 6 combinations of primers. The 6 banding patterns as well as the sequences of the bands extracted were analysed and compared. In addition, to assess the effect of the position of the GC clamp, the melting profiles of the sequences of Aphanizomenon flos-aquae PMC9707 and Synechococcus sp. MH305 were determined, with the GC clamp in the 3' or 5' position. Results showed that the use of two separate amplifications allowed a more complete study of the molecular diversity of the cyanobacterial community investigated. Furthermore, similar richness and identical phylogenetic assignments of extracted bands were obtained irrespective of the positioning of the GC clamp.     

Epilithic cyanobacterial communities of a marine tropical beach rock (Heron Island, Great Barrier Reef): diversity and diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification "microbialite" origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

Determination of cyanobacterial diversity during algal blooms in Daechung Reservoir, Korea, on the basis of cpcBA intergenic spacer region analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.