UNRAVELING MOLECULAR DIVERSITY AND PHYLOGENY OF APHANIZOMENON (NOSTOCALES,CYANOBACTERIA) STRAINS ISOLATED FROM CHINA1

Fifty‐three strains of the genus Aphanizomenon isolated from Chinese waters were employed to conduct morphological examination and sequencing of the 16S rRNA gene, rbcLX (RUBISCO), and cpcBA‐IGS gene regions. Based on morphological characteristics, the examined strains were divided into three morphotypes [Aph. flos‐aquae Bréb. ex Bornet et Flahault, Aph. gracile Lemmerm., and Aph. issatchenkoi (Usacer) Proshk.‐Lavr.]. Phylogenetic analysis based on 16S rRNA and rbcLX showed that Aphanizomenon strains could be divided into three main clades (Clade A of Aph. flos‐aquae, Clade B of Aph. gracile, and Clade C of Aph. issatchenkoi), but two additional clades formed by Aph. ovalisporum and Aph. aphanizomenoides were detected in the 16S rDNA‐based topology. All Aph. issatchenkoi strains contained an additional 175 nucleotides from the 779 to 954 nucleotide location in rbcLX region, compared with strains of Aph. flos‐aquae and Aph. gracile. The cpcBA‐IGS‐based phylogenetic tree revealed that Aph. issatchenkoi strains were not discriminated from Aph. flos‐aquae strains; however, a concatenated alignment of 16S rDNA, rbcLX, and cpcBA‐IGS led to the three distinct clades (Aph. flos‐aquae, Aph. gracile, and Aph. issatchenkoi, respectively). It is suggested that the taxonomic revision of Aphanizomenon and Anabaena genera is required to be performed by employing multilocus sequence analysis and polyphasic studies.     

Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.     

FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.     

Effect of temperature and soluble reactive phosphorus on abundance of Aphanizomenon flos-aquae (Cyanophyceae)

Filament density of Aphanizomenon flos‐aquae (Lemmerm.) Ralfs, water temperature and soluble reactive phosphorus (SRP) were measured from April to August in 1993–1996 in Lake Barato, Hokkaido, Japan. In addition, growth characteristics and internal phosphorus (P) utilization of Aph. flos‐aquae were evaluated under P limitation at three temperatures (15, 20 and 25?C) to clarify the role of internal accumulated P for its growth in the incubation experiment. The filament density was highest in early July 1994, when SRP concentration had not yet decreased and the water temperature was high. These are important factors favoring an increase in abundance of this species in L. Barato. During batch culture, the time course of the stationary phase was shortest at 25?C and longest at 15?C; the cellular C:P molar ratio was 111 under P sufficiency and increased eight‐ to 12‐fold under P limitation. As the C:P ratio was significantly higher in the decreasing phase at 15?C, Aph. flos‐aquae may be more adaptable to Plimitation at 15?C than at 20?C and 25?C. However, the low temperatures did not favor the abundance of Aph. flos‐aquae in 1996. This indicates that the filament density of Aph. flos‐aquae decreases before it reaches the maximum value for some reason under P limitation in L. Barato.     

Solitary terminal cells of Aphanizomenon gracile (Cyanobacteria,Nostocales) can divide and renew trichomes

An attempt was made to find evidence that morphologically distinct terminal cells of filamentous cyanobacterium Aphanizomenon gracile strain CCALA 8 are capable of dividing and forming trichomes. Based on our current knowledge, the division of morphologically diversified terminal cells is possible in nostocalean cyanobacteria. However, this process has been observed only in a few species. Terminal cells of A. gracile differ morphologically from other vegetative cells of a trichome, as they are not hyaline and can sometimes be found as solitary cells in cultures. Hence, it was reasonable for us to suspect that these cells are capable of dividing and forming trichomes. We observed terminal cells under a light and transmission electron microscope. Microscopic observations revealed that the septum formed in both solitary terminal cells and in terminal cells attached to trichomes. Our study is the first to demonstrate division and renewal of trichomes in terminal cells of A. gracile. Previously, such mode of reproduction was described only for another nostocalean cyanobacterium Raphidiopsis mediterranea. Moreover, our findings further emphasize the variability among members that belong to the genus Aphanizomenon , in which a type species (A. flos‐aquae) has hyaline cells incapable of dividing and renewing trichomes, while A. gracile can additionally propagate by solitary terminal cells division. This additional feature distinguishing A. gracile from typical species of Aphanizomenon, such as A. flos‐aquae, might be valuable for resolving taxonomic position of the species considering ambiguous genetic relationship between A. gracile and A. flos‐aquae.     

BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)

The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.     

Polyphasic characterization of water bloom forming Raphidiopsis species (cyanobacteria) from central China

Two strains of Raphidiopsis Fritsch et Rich were isolated from a fishpond in Wuhan city, China and rendered axenic, and characterized by a combination of morphological, physiological, biochemical and genetic methods. Morphologically the strains were identified as Raphidiopsis mediterranea Skuja (straight trichomes) and R. curvata Fritsch et Rich (coiled trichomes). These two strains demonstrated slight differences in optimal temperature range and GC content, while sharing some common characteristics including inability to grow hetertrophically, similar salinity tolerance (up to 0.78%) and an identical fatty acid composition. Cyanotoxins were not found in the strain of R. mediterranea, however, the strain of R. curvata contained both deoxy-cylindrospermopsin and cylindrospermopsin. Phylogenetic affiliations inferred from 16S rRNA gene sequences demonstrated that both Raphidiopsis strains clustered with Cylindrospermopsis, demonstrating their phylogenetic ties to Nostocaceae.     

Phylogenetic identification of three strains of rhizosphere bacteria based on the results of 16s rRNA gene analysis and genetic typing

The rhizosphere nitrogen-fixing bacteria Herbaspirillum frisingense B416, Burkholderia sp. 418, and Herbaspirillum huttiense B601 (degrader of chlorinated s-triazines) were identified by phylogenetic analysis of the 16S rRNA gene sequences, characterization of the 16S–23S intergenic spacer region, Rep-PCR genotyping, and assessment of differentiating phenotypic characteristics. The results obtained indicate that, for correct taxonomic affiliation by comparative analysis of 16S rRNA gene sequences, the ratio between intra-and interspecies variability of these sequences within the group of bacteria closely related to the identified strain should be taken into consideration. If the interspecies differences between 16S rRNA genes are insufficient for differentiation of closely related species, ribotyping and Rep-PCR analysis of genomic DNA can be used for determination of the species affiliation.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

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.     

Seasonal variations in the diel vertical distribution of phytoplankton and zooplankton in a shallow pond

The seasonal succession of phytoplankton diversity, and the variations in the diel vertical distribution of phyto‐ and zooplankton were investigated in a small shallow pond (1.7 m water depth) in 2003. It was inferred that the water tended to stratify weakly in the daytime from February to June. In February and April, the green alga Golenkinia radiata Chodat dominated the phytoplankton assemblage. The cell density of G. radiata greatly decreased in April, when rotifers increased near the bottom. The vertical mixing was attenuated in June, large populations of the euglenoids (Lepocinclis salina Fritsch, Phacus acuminatus Stokes, Trachelomonas hispida (Perty) Stein et Deflandre) developed, and the cyanobacterium Aphanizomenon flos‐aquae var. klebahnii Elenk. appeared at low density. Euglenoids and A. flos‐aquae were mostly distributed in the bottom layer. In late September, when the water was mixed throughout the day, euglenoids and A. flos‐aquae were distributed evenly throughout the water column. The zooplankton (cyclopoid copepods and rotifers) densities in September were the lowest throughout the year. The vertical mixing increased in November, and the phytoplankton community was composed of A. flos‐aquae, P. acuminatus, T. hispida and the green alga Ankistrodesmus falcatus (Corda) Ralfs. In November, at the final stage of water bloom of A. flos‐aquae, its population density decreased with depth. The two euglenoids exhibited similar cell distributions at 0.8 m and 1.6 m during 1–3 November. A. falcatus was distributed evenly throughout the water column; however, when the vertical mixing lessened, the cells at the surface started to sink. Copepod nauplii and rotifers appeared at high densities in November. Seasonal variation in the phytoplankton community structure in the pond seemed to be related to the vertical mixing of the water. In addition, zooplankton, especially rotifers, might play an important role in initiating a spring clear‐water phase and in the bloom collapse of A. flos‐aquae.     

<Emphasis Type="Italic">Halorubrum cibi</Emphasis> sp. nov., an extremely halophilic archaeon from salt-fermented seafood

Strain B31^T is a Gram-staining-negative, motile, and extremely halophilic archaeon that was isolated from salt-fermented seafood. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were determined. Phylogenetic analysis of its 16S rRNA gene sequence and composition of its major polar lipids placed this archaeon in the genus Halorubrum of the family Halobacteriaceae. Strain B31^T showed 97.3, 97.2, and 96.9 % 16S rRNA similarity to the type strains of Halorubrum alkaliphilum, Hrr. tibetense, and Hrr. vacuolatum, respectively. Its major polar lipids were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Genomic DNA from strain B31^T has a 61.7 mol% G+C content. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, identified genotypic and phenotypic differences between strain B31^T and other Halorubrum species. The type strain of the novel species is B31^T (=JCM 15757^T =DSM 19504^T).     

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

A new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.     

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose‐fed laboratory‐scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram‐positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long‐term exposure to tylosin are attributed to the direct inhibition of propionate‐oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH. Biotechnol. Bioeng. 2011;108: 296–305. © 2010 Wiley Periodicals, Inc.     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

Drouetiella epilithica sp. nov. and Drouetiella lurida (Oculatellaceae,Synechococcales) isolated in the Republic of Korea based on the polyphasic approach

Five strains of Drouetiella (ACKU666, 667, 668, 669 and 670) were isolated from gravels in water, stone monument and coastal mudflat in Korea, and were studied using morphological and molecular traits. All five strains had thin and simple trichomes and exhibited false branching. From these strains, four strains (ACKU666, 667, 668 and 669) exhibited similar cell lengths with reddish–brown colored cells such as Drouetiella lurida. The 16S rRNA gene phylogeny showed the four strains formed a clade with Drouetiella lurida, and their DNA similarity was calculated to be 99.1–100%. The color of strain ACKU670 appeared to be in bright blue–green color like Drouetiella fasciculata, and their thylakoids showed a parietal arrangement, which is a characteristic feature of the family Oculatellaceae. Strain ACKU670 turned out to be a sister clade to the D. lurida according to the phylogenetic analysis of the 16S rRNA gene. The 16–23S rRNA internal transcribed spacer secondary folding structure (D1–D1′, Box-B and V3 helices) confirmed the uniqueness of strain ACKU670, therefore indicating differences from the related species. Considering all the results, we described our strain ACKU670 as Drouetiella epilithica sp. nov. in accordance with the International Code of Nomenclature for Algae, Fungi and Plants.     

Anatoxin-a-producing Raphidiopsis mediterranea Skuja var. grandis Hill is one ecotype of non-heterocytous Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. in Japanese lakes

In this study, we sought to clarify the phylogenetic and ecological relationships of anatoxin-a-producing strains of Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. and Raphidiopsis mediterranea Skuja var. grandis Hill in Japanese lakes. We isolated 60 strains from six water bodies and evaluated their ability to produce anatoxin-a and nitrogenase, and determined the sequences of the 16S–23S rDNA internal transcribed spacer region. The results consistently indicated that all R. mediterranea ver. grandis-like strains were non-heterocytous, anatoxin-a-producing C. issatschenkoi. We also evaluated the distribution of anatoxin-a-producing strains of C. issatschenkoi in 88 water bodies using primers and a probe specifically developed in this study for quantitative PCR. By this method, we detected anatoxin-a-producing strains in 16 water bodies, while by automated rRNA intergenic spacer analysis, C. issatschenkoi was detected in 24 water bodies, indicating that the toxic and non-toxic strains occur in different environments. Although C. issatschenkoi has been classified as the functional group H1, which is tolerant to low nitrogen concentrations, the anatoxin-a-producing strains were detected from water bodies with low TOC/TON because of the lack of nitrogen-fixing ability. These results suggest that different genotypes of C. issatschenkoi have different ecotypes and prefer different environments.