MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF PLANKTONIC CYANOBACTERIA FROM BELGIUM AND LUXEMBOURG1

For the first time in Belgium and Luxembourg, the diversity and taxonomy of 95 cyanobacterial strains isolated from freshwater blooms were assessed by the comparison of phenotypes and partial 16S rRNA gene sequences. The results showed the high diversity of nanoplanktonic, picoplanktonic, and benthic–periphytic cyanobacteria accompanying the main bloom‐forming taxa. Indeed, besides 15 morphotypes of bloom‐forming taxa, seven non‐bloom‐forming planktonic morphotypes and 11 morphotypes from benthic–periphytic taxa were isolated in culture from the plankton samples of 35 water bodies. The bloom‐forming strains belonged to the genera Microcystis, Woronichinia, Planktothrix, Anabaena, and Aphanizomenon, whereas the other strains isolated from the same samples were assigned to the nanoplanktonic Aphanocapsa, Aphanothece, Snowella, and Pseudanabaena; to the picoplanktonic Cyanobium; and to the benthic–periphytic Geitlerinema, Komvophoron, Leptolyngbya, Lyngbya, Phormidium, Calothrix, Nostoc, and Trichormus. The results supported both the polyphyletism of genera such as Aphanocapsa, Aphanothece, Leptolyngbya, Geitlerinema, Anabaena, and Aphanizomenon as well as the validity of genera such as Microcystis, Planktothrix, and Pseudanabaena with gas vesicles and cells constricted at the cross wall. The results obtained showed the close relationship between Snowella and Woronichinia for which very few sequences exist. The first sequence of Komvophoron appeared poorly related to other available cyanobacterial sequences. Although in a few cases a good agreement existed between phenotypic and genotypic features, there was generally a discrepancy. Strains with identical morphotypes show small differences in the 16S rRNA sequences, which might be related to the different chemical properties of their habitats. The results showed the importance of the polyphasic approach in order to improve the taxonomy of cyanobacteria.     

Characterization of Cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene

Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera. Received: 16 December 1996 / Accepted: 14 May 1997     

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.     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature. Electronic Publication     

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.     

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia,Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.     

Genotypic and Phenotypic Diversity of Cyanobacteria in Biological Soil Crusts of the Succulent Karoo and Nama Karoo of Southern Africa

Biological soil crusts (BSCs) are communities of cryptogamic organisms, occurring in arid and semiarid regions all over the world. Based on both morphological identification and genetic analyses, we established a first cyanobacterial inventory using the biphasic approach for BSCs within two major biomes of southern Africa. The samples were collected at two different sites in the Succulent Karoo and one in the Nama Karoo. After cultivation and morphological identification, the 16S rRNA gene was sequenced from the cyanobacterial cultures. From the soil samples, the DNA was extracted, and the 16S rRNA gene sequenced. All the sequences of the clone libraries from soil and cultures were compared with those of the public databases. Forty-five different species were morphologically identified in the samples of the Succulent Karoo (observatories of Soebatsfontein and Goedehoop). Based on the genetic analyses, 60 operational taxonomic units (OTUs) were identified for the Succulent Karoo and 43 for the Nama Karoo (based on 95 % sequence similarity). The cloned sequences corresponded well with the morphologically described taxa in cultures and sequences in the public databases. Besides known species of typical crust-forming cyanobacterial genera (Microcoleus, Phormidium, Tolypothrix and Scytonema), we found sequences of so far undescribed species of the genera Leptolyngbya, Pseudanabaena, Phormidium, Oscillatoria, Schizothrix and Microcoleus. Most OTUs were restricted to distinct sites. Grazed soils showed lower taxa numbers than undisturbed soils, implying the presence of early successional crust types and reduced soil surface protection. Our combined approach of morphological identification and genetic analyses allowed both a taxa inventory and the analysis of species occurring under specific habitat conditions.     

Morphological and phylogenetic diversity of thermophilic cyanobacteria in Algerian hot springs

Geothermal springs in Algeria have been known since the Roman Empire. They mainly locate in Eastern Algeria and are inhabited by thermophilic organisms, which include cyanobacteria forming mats and concretions. In this work, we have investigated the cyanobacterial diversity of these springs. Cyanobacteria were collected from water, concretions and mats in nine hot springs with water temperatures ranging from 39 to 93 °C. Samples were collected for isolation in culture, microscopic morphological examination, and molecular diversity analysis based on 16S rRNA gene sequences. Nineteen different cyanobacterial morphotypes were identified, the most abundant of which were three species of Leptolyngbya, accompanied by members of the genera Gloeocapsa, Gloeocapsopsis, Stigonema, Fischerella, Synechocystis, Microcoleus, Cyanobacterium, Chroococcus and Geitlerinema. Molecular diversity analyses were in good general agreement with classical identification and allowed the detection of additional species in three springs with temperatures higher than 50 °C. They corresponded to a Synechococcus clade and to relatives of the intracellularly calcifying Candidatus Gloeomargarita lithophora. The hottest springs were dominated by members of Leptolyngbya, Synechococcus-like cyanobacteria and Gloeomargarita, whereas Oscillatoriales other than Leptolyngbya, Chroococcales and Stigonematales dominated lower temperature springs. The isolation of some of these strains sets the ground for future studies on the biology of thermophilic cyanobacteria.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

铜陵铜尾矿废弃地生物土壤结皮中的蓝藻多样性

生物土壤结皮是生态系统原生演替过程中的一个早期阶段,在铜陵铜尾矿废弃地自然生态恢复过程中生物土壤结皮在尾矿废弃地表面广泛分布.以生长在铜陵杨山冲和铜官山2处铜尾矿废弃地的生物土壤结皮为研究对象,运用常规培养方法和变性梯度凝胶电泳技术(PCR-DGGE)对不同群落生物土壤结皮中的蓝藻多样性及优势类群进行研究.结果表明2种研究方法所获得的蓝藻种类组成具有明显差异.显微观察结果表明常规培养试验中主要蓝藻类群为微囊藻属(Microcystis)、色球藻属(Chroococcus)、颤藻属(Oscillatoria)、念珠藻属(Nostoc)和浮鞘丝藻属(Planktolyngbya),其中优势种类主要为铜绿微囊藻(Microcystis aeruginosa)、断裂颤藻(Oscillatoria fracta)和细浮鞘丝藻(Planktolyngbya subtilis);提取样品中微生物总DNA,对蓝藻16SrRNA进行PCR-DGGE分析,回收DGGE图谱中24个条带进行测序分析,结果显示,所有序列与GenBank数据库中的近缘蓝藻的相似性系数均在93％以上,其中优势蓝藻类群主要隶属于微鞘藻属(Microcoleus)和细鞘丝藻属(Leptolyngbya),裸地(YL)处和木贼群落下尾矿表面(YM)的生物土壤结皮中优势蓝藻类群主要为微鞘藻属,而黄色真藓-藻类混合结皮(YT)和白茅群落( YB,TG)下的生物土壤结皮中的优势类群主要隶属于细鞘丝藻属.     

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.     

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Benthic cyanobacterial communities from Guadarrama River (Spain) biofilms were examined using temperature gradient gel electrophoresis (TGGE), comparing the results with microscopic analyses of field-fixed samples and the genetic characterization of cultured isolates from the river. Changes in the structure and composition of cyanobacterial communities and their possible association with eutrophication in the river downstream were studied by examining complex TGGE patterns, band extraction, and subsequent sequencing of 16S rRNA gene fragments. Band profiles differed among sampling sites depending on differences in water quality. The results showed that TGGE band richness decreased in a downstream direction, and there was a clear clustering of phylotypes on the basis of their origins from different locations according to their ecological requirements. Multivariate analyses (cluster analysis and canonical correspondence analysis) corroborated these differences. Results were consistent with those obtained from microscopic observations of field-fixed samples. According to the phylogenetic analysis, morphotypes observed in natural samples were the most common phylotypes in the TGGE sequences. These phylotypes were closely related to Chamaesiphon, Aphanocapsa, Pleurocapsa, Cyanobium, Pseudanabaena, Phormidium, and Leptolyngbya. Differences in the populations in response to environmental variables, principally nutrient concentrations (dissolved inorganic nitrogen and soluble reactive phosphorus), were found. Some phylotypes were associated with low nutrient concentrations and high levels of dissolved oxygen, while other phylotypes were associated with eutrophic-hypertrophic conditions. These results support the view that once a community has been characterized and its genetic fingerprint obtained, this technique could be used for the purpose of monitoring rivers.     

Diversity and bioactivities of nostocacean cyanobacteria isolated from paddy soil in Vietnam

Nostocacean cyanobacteria are one of the important components of paddy fields due to their ability to fix atmospheric nitrogen and supply phytohormones for crop growth. In this study, 13 Nostoc strains isolated from paddy soils in Vietnam were classified using a polyphasic approach. The results showed a high diversity of the isolated strains that represented seven morphotypes corresponding to five genotypes, with 16S rRNA gene sequence similarity values ranging between 94.97–99.78% compared to the available sequences from GenBank. Bioassay assessment revealed that 11 out of 13 strains possessed antibacterial activities, three of which exhibited cytotoxic activities on MCF7 and HCT116 cells with an IC₅₀ ranging from 47.8 μg mL^?1 to 232.0 μg mL^?1. Interestingly, strains with identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activity profiles.     

Novel Cyanobacterial Diversity Found in Costa Rican Thermal Springs Associated with Rincon de la Vieja and Miravalles Volcanoes: A Polyphasic Approach

Central America is one of the most important biodiversity hot spots in the world, and Costa Rican microbial communities from thermal springs are the best characterized in the isthmus. Miravalles is an inactive quaternary stratovolcano, and the Rincón de la Vieja is a unique active volcano, in whose slopes diverse hydrothermal springs, such as Las Lilas, are located. These springs harbor extensive microbial mats, whose diversity has been studied. Based on their importance as primary producers, in this study we focused on cultured cyanobacterial diversity from two geothermal environments of northern Costa Rica. Several cultural, molecular and taxonomic techniques were employed to maximize the results of a polyphasic approach. Sample collection sites were physicochemically described, and strains were isolated and characterized by light and electron microscopy. Phylogenetic analysis was performed using 16S rRNA gene sequences and amplified ribosomal DNA restriction analysis (ARDRA). Fifty‐six phylotypes were isolated and classified into 21 morphotypes and identified in 14 genera, some of them might be new species within these genera. Furthermore, according to phylogenetic analysis, there are three possible new genera in our collection. Miravalles and Las Lilas thermal springs are reservoirs of novel phylogeographic lineages of phototrophic microorganisms. This study is the first report of strains that belong to the genera Gloeocapsa, Stanieria, Microseira, Klisinema and Oculatella isolated from thermal springs and growing at temperatures above 50°C. We also obtained isolates assigned to Synechococcus, Leptolyngbya spp., and Fischerella, which are considered typical strains in these environments.     

海南西海岸真红树内生细菌多样性及其延缓衰老活性研究

该文设计9种分离培养基,采用稀释涂布法从14份真红树植物的46份组织样品中分离纯化内生细菌。并基于菌株形态学特征和16S rRNA基因序列确定分离菌株的种属及分析其物种多样性,采用秀丽隐杆线虫模型筛选菌株延缓衰老活性。结果表明:(1)通过基因序列去重复后从46份真红树植物组织样品中获得32株海洋细菌,基于菌株16S rRNA基因序列信息分析,覆盖12科17属,其中芽孢杆菌属(Bacillus)为优势菌属,并获得1株疑似橙单胞菌属(Aurantimonas)新种,16S rRNA基因序列相似性低于97%;(2)经过秀丽隐杆线虫粗筛发现3株海洋细菌具有显著延缓线虫衰老的活性(P<0.05)。以上结果表明海南西海岸真红树内生细菌具有物种多样性,部分菌株具有延缓线虫衰老活性。     

Polyphasic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama river (Spain): morphological,molecular, and ecological approaches1

The occurrence and environmental factors responsible for the distribution of benthic cyanobacteria in running waters remain largely unexplored in comparison with those of other aquatic ecosystems. In this study, combined data of ecological characteristics, molecular analysis (based on 16S rRNA gene), and direct microscopic inspection of environmental samples were analyzed in parallel with the morphological characterization of the isolated strains to investigate benthic cyanobacterial diversity in the Guadarrama river (Spain). A total of 17 species were identified that belonged to the genera Aphanocapsa, Pleurocapsa, Chroococcus, Chamaesiphon, Cyanobium, Pseudan‐abaena, Leptolyngbya, Phormidium, Nostoc, and Tolypothrix. Phenotypic features were associated with the results of 16S rRNA gene sequencing, complementing existing morphological and genetic databases. A decrease in the cyanobacterial diversity was observed along a pollution gradient in the river. Water quality differed among the sampling sites, and variation in nutrient content was the principal difference among locations. These characteristics were closely associated with an upstream‐downstream eutrophic gradient. Canonical correspondence analysis distinguished three groups of species with respect to the eutrophication gradient. The first group (Tolypothrix cf. tenuis, Nostoc punctiforme, Nostoc piscinale, Chamaesiphon investiens, Chroococcus minor, Leptolyngbya nostocorum, and Leptolyngbya tenuis) was characteristic of waters with low levels of nutrients. The second group (Cyanobium sp., Chamaesiphon polymorphus, Leptolyngbya boryana, Phormidium autumnale, Phormidium sp., and Aphanocapsa cf. rivularis) was characteristic of polluted waters, its members appearing mainly in great abundance under eutrophic‐hypertrophic conditions. The third group of species (Pseudanabaena catenata, Aphanocapsa muscicola, and Nostoc carneum) was present at upstream and downstream sites.     

Cytomorphological and Genetic Characterization of Troglobitic Leptolyngbya Strains Isolated from Roman Hypogea

Six Leptolyngbya strains, isolated from the archaeological surfaces of hypogean sites, were phenotypically and genetically characterized by light and electron microscopy and 16S rRNA gene and 16S-23S internally transcribed spacer (ITS) sequencing. Three phycoerythrin-rich (red) and three phycocyanin-rich (green) isolates were assigned to different operational taxonomic units (OTUs). Among the green isolates, one strain showed an OTU intraspecific variation due to differences in the ITS sequences and genomic polymorphism. Within the ITS sequence, variable regions, conserved domains and tRNA^Ile and tRNA^Ala genes showed high sequence identity among the phylotypes. Together, these data indicated a relatedness of the six strains to other Leptolyngbya from subaerophytic and geothermal environments and allowed the definition of novel Leptolyngbya OTUs.     

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

Background

Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.

Methods

The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.

Results

Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.

Conclusion

Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella.