Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.     

Development of phoH as a novel signature gene for assessing marine phage diversity

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment.     

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

The community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (?94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus ‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus ‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.     

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.     

Composition and diversity of nifH genes of nitrogen-fixing cyanobacteria associated with boreal forest feather mosses

Recent studies have revealed that nitrogen fixation by cyanobacteria living in association with feather mosses is a major input of nitrogen to boreal forests. We characterized the community composition and diversity of cyanobacterial nifH phylotypes associated with each of two feather moss species (Pleurozium schreberi and Hylocomium splendens) on each of 30 lake islands varying in ecosystem properties in northern Sweden. Nitrogen fixation was measured using acetylene reduction, and nifH sequences were amplified using general and cyanobacterial selective primers, separated and analyzed using density gradient gel electrophoresis (DGGE) or cloning, and further sequenced for phylogenetic analyses. Analyses of DGGE fingerprinting patterns revealed two host-specific clusters (one for each moss species), and sequence analysis showed five clusters of nifH phylotypes originating from heterocystous cyanobacteria. For H. splendens only, N(2) fixation was related to both nifH composition and diversity among islands. We demonstrated that the cyanobacterial communities associated with feather mosses show a high degree of host specificity. However, phylotype composition and diversity, and nitrogen fixation, did not differ among groups of islands that varied greatly in their availability of resources. These results suggest that moss species identity, but not extrinsic environmental conditions, serves as the primary determinant of nitrogen-fixing cyanobacterial communities that inhabit mosses.     

基于克隆文库法研究古尔班通古特沙漠蓝藻多样性

该研究采用克隆文库法研究古尔班通古特沙漠藻类生物结皮中蓝藻多样性及分布.在古尔班通古特沙漠不同区域采集10份藻类结皮代表性土样,分别构建古尔班通古特沙漠蓝藻16S rRNA和psbA基因克隆文库,并进行系统发育分析,对蓝藻多样性和丰度与环境因子进行关联分析,研究蓝藻分布特点及影响因子.结果 显示:(1)16S rRNA...     

Spatially differing bacterial communities in water columns of the northern Baltic Sea

The Baltic Sea is a large, shallow, and strongly stratified brackish water basin. It suffers from eutrophication, toxic cyanobacterial blooms, and oxygen depletion, all of which pose a threat to local marine communities. In this study, the diversity and community structure of the northern Baltic Sea bacterial communities in the water column were, for the first time, thoroughly studied by 454 sequencing. The spring and autumn bacterial communities were one order of magnitude less diverse than those in recently studied oceanic habitats. Patchiness and strong stratification were clearly detectable; <1% of operational taxonomic units were shared among 11 samples. The community composition was more uniform horizontally (at a fixed depth) between different sites than vertically within one sampling site, implying that the community structure was affected by prevailing physical and hydrochemical conditions. Taxonomic affiliations revealed a total of 23 bacterial classes and 169 genera, while 5% of the sequences remained unclassified. The cyanobacteria accounted for <2% of the sequences, and potentially toxic cyanobacterial genera were essentially absent during the sampling seasons.     

Characterization of Trichodesmium spp. by genetic techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

Cyanobacterial diversity and activity in modern conical microbialites

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high‐resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non‐heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 μm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.     

Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.     

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Low cyanobacterial diversity in biotopes of the Transantarctic Mountains and Shackleton Range (80-82°S), Antarctica

The evolutionary history and geographical isolation of the Antarctic continent have produced a unique environment rich in endemic organisms. In many regions of Antarctica, cyanobacteria are the dominant phototrophs in both aquatic and terrestrial ecosystems. We have used microscopic and molecular approaches to examine the cyanobacterial diversity of biotopes at two inland continental Antarctic sites (80-82°S). These are among the most southerly locations where freshwater-related ecosystems are present. The results showed a low cyanobacterial diversity, with only 3-7 operational taxonomic units (OTUs) per sample obtained by a combination of strain isolations, clone libraries and denaturing gradient gel electrophoresis based on 16S rRNA genes. One OTU was potentially endemic to Antarctica and is present in several regions of the continent. Four OTUs were shared by the samples from Forlidas Pond and the surrounding terrestrial mats. Only one OTU, but no internal transcribed spacer (ITS) sequences, was common to Forlidas Pond and Lundstr?m Lake. The ITS sequences were shown to further discriminate different genotypes within the OTUs. ITS sequences from Antarctic locations appear to be more closely related to each other than to non-Antarctic sequences. Future research in inland continental Antarctica will shed more light on the geographical distribution and evolutionary isolation of cyanobacteria in these extreme habitats.     

Diurnal variations in photosynthetic rates: comparisons of ultraphytoplankton with a larger phytoplankton size fraction

We compared the composition and photosynthetic activity of twosize fractions of phytoplankton during a cruise from the Gulfof Maine to the Sargasso Sea in August 1983. At every station,and at every depth, ultraplankton (defined here as cells passingthrough 3 µm pores in filters) made a major contributionto both the standing crop of chlorophyll and the rate of primaryproduction. Ultraphytoplankton assemblages were dominated byphycoerythrin-rich cyanobacteria. Overall, the ultraplanktoncontribution to total primary production was greatest at lowphoton fluxes: (i) at the beginning and end of the photoperiod;(ii) with increasing depth in the euphotic zone; and (iii) whendaily irradiance was low. The composition of ultraphytoplanktonvaried with depth. Surface (2 m) ultraphytoplankton assemblageswere almost exclusively composed of phycoerythrin-rich cyanobacteriawith smaller (0.2–0.8 µm) cyanobacteria predominating.Below the surface mixed-layer, the proportion of larger (0.8–30 µm) to smaller cyanobacteria increased and the eukaryoticcomponent of the ultraphytoplankton often became important.At two Sargasso Sea stations, the greatest numbers of cyanobacteriawere below the mixed layer at the 1% light level, while themaximum numbers of eukaryotic ultraphytoplankters occurred deeperstill, at the 0.5% light level, coincident with the chlorophyllmaximum. At the bottom of the euphotic layer in the SargassoSea. eukaryotes numerically dominated the ultraphytoplanktonand made a major contribution to the chlorophyll maximum.     

Cyanobacterial evolution: results of 16S ribosomal ribonucleic acid sequence analyses.

We report here the sequences of oligonucleotides released by T1-ribonuclease digestion of the 16S ribosomal RNA's (rRNA's) of unicellular cyanobacteria Agmenellum quadruplicatum (strain BG-1) and Synechococcus 7502. We compare them with sequences previously obtained for the 16S RNA's of six other cyanobacteria and two chloroplasts, and conclude that: (i) Synechocystis-like unicells form a discrete cluster which also (and surprisingly) includes Agmenelium quadruplicatum, usually considered to be a Synechococcus; (ii) filamentous cyanobacteria of the genera Nostoc and Fischerella arose from within the Synechocystis group; (iii) phylogenetic diversity (and hence presumably evolutionary antiquity) within the Synechococcus group is very great; and (iv) red algal chloroplasts are of definite cyanobacterial origin, while Euglena chloroplasts are of separate and quite possibly noncyanobacterial origin. We also present the results of a computer-aided search among the 10 oligonucleotide 'catalogues' for families of related but nonidentical sequences. Examination of these families reinforces the above conclusions.     

Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans.

The extent of the diversity of marine prokaryotes is not well known, primarily because of poor cultivability. However, new techniques permit the characterization of such organisms without culturing, via 16S rRNA sequences obtained directly from biomass. We performed such an analysis by polymerase chain reaction amplification with universal primers on five oligotrophic open-ocean samples: from 100-m (three samples) and 500-m depths in the western California Current (Pacific Ocean) and from a 10-m depth in the Atlantic Ocean near Bermuda. Of 61 clones, 90% were in clusters of two or more related marine clones obtained by ourselves or others. We report 15 clones related to clone SAR 11 found earlier near Bermuda (S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field, Nature [London] 345:60-63, 1990), 11 related to marine cyanobacteria, 9 clustered in a group affiliated with gram-positive bacteria, 9 in an archaeal cluster we recently described (mostly from the 500-m sample), 4 in a novel gamma-proteobacterial cluster, and 6 in three two-membered clusters (including other archaea). One clone was related to flavobacteria. Only the cyanobacteria plus one other clone, related to Roseobacter denitrificans (formerly Erythrobacter longus Och114), were within 10% sequence identity to any previously sequenced cultured organism in a major data base. We never found more than two occurrences of the same sequence in a sample, although four times we found identical sequences between samples, two of which were between oceans; one of these sequences was also identical to SAR 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Targeted deep sequencing reveals high diversity and variable dominance of bloom-forming cyanobacteria in eutrophic lakes

Cyanobacterial blooms in eutrophic lakes are severe environmental problems worldwide. To characterize the spatiotemporal heterogeneity of cyanobacterial blooms, a high-throughput method is necessary for the specific detection of cyanobacteria. In this study, the cyanobacterial composition of three eutrophic waters in China (Taihu Lake, Dongqian Lake, and Dongzhen Reservoir) was determined by pyrosequencing the cpcBA intergenic spacer (cpcBA-IGS) of cyanobacteria. A total of 2585 OTUs were obtained from the normalized cpcBA-IGS sequence dataset at a distance of 0.05. The 238 most abundant OTUs contained 92% of the total sequences and were classified into six cyanobacterial groups. The water samples of Taihu Lake were dominated by Microcystis, mixed Nostocales species, Synechococcus, and unclassified cyanobacteria. Besides, all the samples from Taihu Lake were clustered together in the dendrogram based on shared abundant OTUs. The cyanobacterial diversity in Dongqian Lake was dramatically decreased after sediment dredging and Synechococcus became exclusively dominant in this lake. The genus Synechococcus was also dominant in the surface water of Dongzhen Reservoir, while phylogenetically diverse cyanobacteria coexisted at a depth of 10 m in this reservoir. In summary, targeted deep sequencing based on cpcBA-IGS revealed a large diversity of bloom-forming cyanobacteria in eutrophic lakes and spatiotemporal changes in the composition of cyanobacterial communities. The genus Microcystis was the most abundant bloom-forming cyanobacteria in eutrophic lakes, while Synechococcus could be exclusively dominant under appropriate environmental conditions.