Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq ( https://zenodo.org/record/7569105 ), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.     

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: a polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N(2)-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.     

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.      

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus <Emphasis Type="Italic">Phormidium</Emphasis> (Oscillatoriales) from different habitats and geographical sites

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.     

Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies <Emphasis Type="Italic">Phormidium </Emphasis><Emphasis Type="Italic">autumnale</Emphasis> (Oscillatoriales)

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.     

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.     

A bridge too far in naming species: a total evidence approach does not support recognition of four species in Desertifilum (Cyanobacteria)

A population of Desertifilum (Cyanobacteria, Oscillatoriales) from an oligotrophic desertic biotope was isolated and characterized using a polyphasic approach including molecular, morphological, and ecological information. The population was initially assumed to be a new species based on ecological and biogeographic separation from other existing species, however, phylogenetic analyses based on sequences of the 16S rRNA gene and 16S–23S ITS region, placed this strain clearly within the type species, Desertifilum tharense. Comparative analysis of morphology, 16S rRNA gene similarity, 16S–23S ITS secondary structure, and percent dissimilarity of the ITS regions for all characterized strains supports placing the six Desertifilum strains (designated as PD2001/TDC17, UAM‐C/S02, CHAB7200, NapGTcm17, IPPAS B‐1220, and PMC 872.14) into D. tharense. The recognition of Desertifilum salkalinema and Desertifilum dzianense is not supported, although our analysis does support continued recognition of Desertifilum fontinale. Pragmatic criteria for recognition of closely related species are proposed based on this study and others, and more rigorous review of future taxonomic papers is recommended.     

Gene Sequence Phylogenies of the Family <Emphasis Type="Italic">Microbacteriaceae</Emphasis>

The type strains of 32 species of 13 genera of the family Microbacteriaceae were analysed with respect to gene-coding phylogeny for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). The resulting gene trees were compared with the 16S rRNA gene phylogeny of the same strains. The topology of neighbour-joining and maximum parsimony phylogenetic trees, based on nucleic-acid sequences and protein sequences of housekeeping genes, differed from one another, and no gene tree was identical to that of the 16S rRNA gene tree. Most genera analysed containing >1 strain formed phylogenetically coherent taxa. The three pathovars of Curtobacterium flaccumfaciens clustered together to the exclusion of the type strains of other Curtobacterium species in all DNA - and protein-based analyses. In no tree did the distribution of a major taxonomic marker, i.e., diaminobutyric acid versus lysine and/or ornithine in the peptidoglycan, or acyl type of peptidoglycan, correlate with the phylogenetic position of the organisms. The changing phylogenetic position of Agrococcus jenensis was unexpected: This strain defined individual lineages in the trees based on 16S rRNA and gyrB and showed identity with Microbacterium saperdae in the other three gene trees.     

古尔班通古特沙漠生物结皮微鞘藻分类学研究

实验研究了从古尔班通古特沙漠生物土壤结皮中分离纯化培养出的11株与微鞘藻(Microcoleus)形态接近的丝状蓝藻,通过形态特征、16S rRNA和ITS二级结构相结合的多相分析方法对其进行分类学研究。研究结果表明,实验藻株隶属于微鞘藻科(Microcoleaceae)的微鞘藻属(Microcoleus)和束脉藻属(Symplocastrum),其中包括2个中国新记录种:斯坦微鞘藻(Microcoleus steenstrupii)和细长束脉藻(Symplocastrum flechtnerii),另外还有具鞘微鞘藻(Microcoleus vaginatus)和类似斯坦微鞘藻的存疑物种。藻丝多少与排列方式、细胞大小与末端细胞形状,以及16S rRNA系统发育位置是确定微鞘藻(Microcoleus)与束脉藻(Symplocastrum)属于不同物种的关键依据, ITS二级结构是区分属内不同物种的重要参考。     

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.     

cpcBA-IGS as an effective marker to characterize Microcystis wesenbergii (Komárek) Komárek in Kondrateva (cyanobacteria)

Microcystis wesenbergii (Komárek) Komárek in Kondrateva, a major bloom forming cyanobacterial species, possesses unique colonial characteristics which can be easily distinguished from other Microcystis species. However, there is still no genetic marker to effectively characterize M. wesenbergii. In this research, thirteen strains of M. wesenbergii, collected from eight locations in Chinese water bodies were examined for molecular characterization of both cpcBA-IGS sequences (phycocyanin intergenic spacer and flanking regions) and ITS sequences (internal transcribed spacer region between 16S and 23S rDNA). The phylogenetic analysis based on cpcBA-IGS sequences showed that the M. wesenbergii strains formed a distinct cluster with high support values, indicating the cpcBA-IGS region could be used to characterize and distinguish M. wesenbergii from other species of Microcystis. These developed primers were verified to be effective in distinguishing M. wesenbergii from other species of Microcystis and from other species in different genera of cyanobacteria.     

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea)

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.     

Molecular phylogenetics and ecological diversification of the transisthmian fish genus Centropomus (Perciformes: Centropomidae).

Phylogenetic relationships among the 12 recognized fish species in the New World genus Centropomus (Pisces, Centropomidae) were analyzed using allozyme electrophoresis and 618 bp of the mitochondrial DNA 16S ribosomal RNA (rRNA) gene. Molecular phylogenetic trees were generally consistent with previously published partial hypotheses based on morphological evidence. However, previously undefined sister group relationships between major species groups were resolved using molecular data, and phylogenetic hypotheses for Centropomus based on 16S rRNA sequences were better supported than were allozyme-based hypotheses. The high level of congruence among the trees inferred from the nuclear and mitochondrial characters provided a firm phylogenetic basis for analysis of ecological diversification and molecular evolution in the genus. Compared to basal Centropomus species, members of the most nested species group were significantly larger in body size and occupied a marine niche only peripherally utilized by their congeners. We also observed substitution rate heterogeneity among 16S rRNA lineages; in contrast to expectations based on "metabolic rate" and "generation interval" models, relative substitution rates were faster than expected for the group of large-bodied snooks. Using the Pliocene rise of the Central American isthmian marine barrier to calibrate rates of 16S ribosomal gene evolution in Centropomus, we found that the rates for the genus were similar to those reported for higher vertebrates. Analysis of the three sets of transisthmian geminate taxa in Centropomus indicated that two of the pairs were probably formed during the Pliocene rise of the isthmus while the third pair diverged several million years earlier.     

Phylogenetical relationship based on groE genes among phenotypically related Enterobacter,Pantoea, Klebsiella,Serratia and Erwinia species

In an attempt to define the phylogenetical relationship among 17 phenotypically related species of genera Enterobacter, Pantoea, Serratia, Klebsiella and Erwinia, we determined almost all of their groE operon sequences using the polymerase chain reaction direct sequencing method. The number of nucleotide substitutions per site was 0.12+/-0.030. The value was 3.6-fold higher than that of 16S rDNA. As a result, we were successful in constructing molecular phylogenetic trees which had a finer resolution than that based on the 16S rDNA sequences. The phylogenetic trees based on the nucleotide sequences and deduced amino acid sequences of groE operons indicated that the members of genera Enterobacter, Pantoea and Klebsiella were closely related to each other, while Serratia and Erwinia species except Erwinia carotovora, made distinct clades. The close relationship between Enterobacter aerogenes and Klebsiella pneumoniae, that had been suggested by biochemical tests and DNA hybridization, was also supported by our molecular phylogenetic trees.     

台湾海峡鱼类绦虫的分子系统学研究

利用DNA测序技术对台湾海峡部分鱼类绦虫的16S rRNA和18S rRNA基因片段序列进行了分析。使用PAUP4·0b10软件构建的进化树显示,目前关于绦虫二叶目、锥吻目、假叶目、盘头目和四叶目的划分是比较合理的,绦虫进化基本遵循了头节形态从简单到复杂的进化规律。报道了国内首次发现的双叶目绦虫,进化树结果初步支持了巨槽属和棘头属的划分。此外,结果也支持了前孔属绦虫的分类地位。但是,对耳槽属绦虫与阶室属绦虫的形态学划分与分子系统学相矛盾,利用16S rRNA基因对盘头目各种的进化树分析与形态学差异很大,这些问题都需要更多研究来进行深入分析。     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries

Natto-like fermented soybean products are manufactured and consumed in many Asian countries. In this study, we isolated thirty-four Bacillus strains capable of producing gamma-polyglutamic acid (PGA) from natto in mountainous areas of South Asia and Southeast Asia and from soils in Japan. To elucidate the phylogeny of these PGA-producing strains, phylogenetic trees based on sequences of 16S rDNA, housekeeping genes of rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) were constructed. A phylogenetic tree based on 16S rDNA sequences showed that twenty-one isolates were clustered in the same group of B. subtilis. The other thirteen isolates were located in the cluster of B. amyloliquefaciens. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. The results of the present study indicate that PGA-producing strains isolated from local natto in Asian countries and soil in Japan can be divided into two species, B. subtilis and B. amyloliquefaciens.     

Comparative phylogeny of the ammonia monooxygenase subunit A and 16S rRNA genes of ammonia-oxidizing bacteria

A fragment of the ammonia monooxygenase gene (amoA) from 31 strains of ammonia-oxidizing bacteria (AOB) was sequenced and analysed phylogenetically. The results were compared with the phylogeny of 16S rDNA from AOB. For most groups of AOB we found a high consistency between the phylogenetic trees based on the 16S rDNA and amoA sequences. Although it is not a phylogenetic marker, using the amoA as a probe when studying microbial diversity will probably reduce the amount of non-AOB detected, compared to using rDNA based probes. The data presented in this paper extend and improve the basis for application of amoA in studies of AOB in the environment.     

A Natural View of Microbial Biodiversity within Hot Spring Cyanobacterial Mat Communities

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.