Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Seasonal variations in virus-host populations in Norwegian coastal waters: focusing on the cyanophage community infecting marine Synechococcus spp

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 x 10(4) Synechococcus cells ml(-1) and 7.2 x 10(3) cyanophage ml(-1). By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.     

Prevalence of Viral Photosynthetic and Capsid Protein Genes from Cyanophages in Two Large and Deep Perialpine Lakes

Cyanophages are important components of aquatic ecosystems, but their genetic diversity has been little investigated in freshwaters. A yearlong survey was conducted in surface waters of the two largest natural perialpine lakes in France (Lake Annecy and Lake Bourget) to investigate part of this cyanophage diversity through the analysis of both structural (e.g., g20) and functional (e.g., psbA) genes. We found that these cyanophage signature genes were prevalent throughout the year but that the community compositions of g20 cyanomyoviruses were significantly different between the two lakes. In contrast, psbA-containing cyanophages seemed to be more similar between the two ecosystems. We also found that a large proportion of g20 sequences grouped with cyanomyophage isolates. psbA sequences, belonging to phages of Synechococcus spp., were characterized by distinct triplet motifs (with a novel viral triplet motif, EFE). Thus, our results show that cyanophages (i) are a diverse viral community in alpine lakes and (ii) are clearly distinct from some other freshwater and marine environments, suggesting the influence of unique biogeographic factors.     

Spatial and temporal changes of cyanophage communities in paddy field soils as revealed by the capsid assembly protein gene g20

Bacteriophages are ubiquitous in various environments. Our previous study revealed the diversity of the cyanophage community in paddy floodwater. In this study, the phylogeny and genetic diversity of cyanophage communities in paddy field soils were reported. The viral capsid assembly protein gene (g20) of cyanophage was amplified with the primers CPS1 and CPS8 from soil DNA extracted during two different sampling times at three sampling sites in Japan. The sequencing results indicated that about 93% of the clones were g20 genes. In total, 70 clones of g20 genes were obtained in this study, of which 69 clones were of cyanophage origin. As evaluated by g20 sequence assemblages in paddy field soils, the unifrac analyses results indicated that cyanophage communities changed among the sampling sites and times and differed from those communities detected in paddy floodwater. The phylogenetic analysis showed that the g20 sequences in paddy field soils were very diverse and distributed into Clusters α, β and ?, as well as four newly formed clusters. Within Clusters β and ?, four unique subclusters were formed from the g20 clones that were only observed in this study. These findings suggested that the cyanophage communities in paddy field soils are different from those found in freshwater, marine water and paddy floodwater.     

The Physical Environment Affects Cyanophage Communities in British Columbia Inlets

Little is known about the natural distribution of viruses that infect the photosynthetically important group of marine prokaryotes, the cyanobacteria. The current investigation reveals that the structure of cyanophage communities is dependent on water column structure. PCR was used to amplify a fragment of the cyanomyovirus gene (g) 20, which codes for the portal vertex protein. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified g20 gene fragments was used to examine variations in cyanophage community structure in three inlets in British Columbia, Canada. Qualitative examination of denaturing gradient gels revealed cyanophage community patterns that reflected the physical structure of the water column as indicated by temperature and salinity. Based on mobility of PCR fragments in the DGGE gels, some cyanophages appeared to be widespread, while others were observed only at specific depths. Cyanophage communities within Salmon Inlet were more related to one another than to communities from either Malaspina Inlet or Pendrell Sound. As well, surface communities in Malaspina Inlet and Pendrell Sound were different when compared to communities at depth. In the same two locations, distinct differences in community composition were observed in communities that coincided with depths of high chlorophyll fluorescence. The observed community shifts over small distances (only a few meters in depth or inlets separated by less than 100 km) support the idea that cyanophage communities separated by small spatial scales develop independently of each other as a result isolation by water column stratification or land mass separation, which may ultimately lead to changes in the distribution or composition of the host community. Present address (S.M. Short): Ocean Sciences Department, University of California, Santa Cruz 1156 High Street Santa Cruz, CA 95064; sshort@es.ucsc.edu     

Seasonal Variations in Virus-Host Populations in Norwegian Coastal Waters: Focusing on the Cyanophage Community Infecting Marine Synechococcus spp.

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 × 10⁴ Synechococcus cells ml⁻¹ and 7.2 × 10³ cyanophage ml⁻¹. By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.     

Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.     

Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.     

Pollution Impacts on Bacterioplankton Diversity in a Tropical Urban Coastal Lagoon System

Despite a great number of published studies addressing estuarine, freshwater and marine bacterial diversity, few have examined urban coastal lagoons in tropical habitats. There is an increasing interest in monitoring opportunistic pathogens as well as indigenous microbial community members in these water bodies by current molecular and microbiological approaches. In this work, bacterial isolates were obtained through selective plate dilution methods to evaluate antibiotic resistances. In addition, 16S rRNA gene libraries were prepared from environmental waters and mixed cultures grown in BHI medium inoculated with Jacarepaguá lagoon waters. Denaturing gradient gel electrophoresis (DGGE) analyses showed distinct community profiles between environmental communities from each studied site and their cultured counterparts. A total of 497 bacterial sequences were analyzed by MOTHUR, yielding 245 operational taxonomic units (OTUs) grouped at 97% similarity. CCA diagrams showcased how several environmental variables affect the distribution of 18 bacterial orders throughout the three distinct habitats. UniFrac metrics and Venn diagrams revealed that bacterial communities retrieved through each experimental approach were significantly different and that only one OTU, closely related to Vibrio cholerae, was shared between them. Potentially pathogenic bacteria were isolated from most sampled environments, fifty percent of which showed antibiotic resistance.     

Genetic diversity and temporal variation in the cyanophage community infecting marine Synechococcus species in Rhode Island's coastal waters

The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10(4) phage ml(-1) in the summer months to less then 10(2) phage ml(-1) during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of Synechococcus spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g., Podoviridae or Siphoviridae) may be important components of the Rhode Island cyanophage community.     

Genetic Diversity and Temporal Variation in the Cyanophage Community Infecting Marine Synechococcus Species in Rhode Island's Coastal Waters

The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10⁴ phage ml⁻¹ in the summer months to less then 10² phage ml⁻¹ during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of Synechococcus spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g., Podoviridae or Siphoviridae) may be important components of the Rhode Island cyanophage community.     

Microbial community composition of anoxic marine sediments in the Bay of Cádiz (Spain)

The composition of the microbial community inhabiting the anoxic coastal sediments of the Bay of Cádiz (southern Spain) was investigated using a molecular approach consisting of PCR cloning and denaturing gradient gel electrophoresis (DGGE), based on 16S rRNA sequences. The total cell count was 1-5 × 10? cells/g sediment and, as determined by catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH), the proportion of Bacteria to Archaea was about 70:30. The analysis of 16S-rRNA gene sequences revealed a wide spectrum of microorganisms, which could be grouped into 111 operational taxonomic units (OTUs). Many of the OTUs showed high phylogenetic similarity to microorganisms living in marine sediments of diverse geographic origin. The phylogenetic groups that were predominantly detected were Firmicutes, Deltaproteobacteria, and Gammaproteobacteria, accounting for 23, 15, and 14% of the clones, respectively. Diversity in the domain Archaea was significantly lower than in the domain Bacteria. The majority of the archaeal OTUs belonged to the Crenarchaeota phylum. Since most of the sequences could not be identified precisely at the genus/species level, the functional roles of the microorganisms in the ecosystem could not be inferred. However, seven OTUs affiliated with the Delta- and Epsilonproteobacteria were identified down to the genus level, with all of the identified genera known to occur in sulfate-rich marine environments.     

Influence of salinity on bacterioplankton communities from the Brazilian rain forest to the coastal Atlantic Ocean

Background

Planktonic bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems, however, the taxa that make up these communities are poorly known. The aim of this study was to investigate bacterial communities in aquatic ecosystems at Ilha Grande, Rio de Janeiro, Brazil, a preserved insular environment of the Atlantic rain forest and how they correlate with a salinity gradient going from terrestrial aquatic habitats to the coastal Atlantic Ocean.

Methodology/Principal Findings

We analyzed chemical and microbiological parameters of water samples and constructed 16S rRNA gene libraries of free living bacteria obtained at three marine (two coastal and one offshore) and three freshwater (water spring, river, and mangrove) environments. A total of 836 sequences were analyzed by MOTHUR, yielding 269 freshwater and 219 marine operational taxonomic units (OTUs) grouped at 97% stringency. Richness and diversity indexes indicated that freshwater environments were the most diverse, especially the water spring. The main bacterial group in freshwater environments was Betaproteobacteria (43.5%), whereas Cyanobacteria (30.5%), Alphaproteobacteria (25.5%), and Gammaproteobacteria (26.3%) dominated the marine ones. Venn diagram showed no overlap between marine and freshwater OTUs at 97% stringency. LIBSHUFF statistics and PCA analysis revealed marked differences between the freshwater and marine libraries suggesting the importance of salinity as a driver of community composition in this habitat. The phylogenetic analysis of marine and freshwater libraries showed that the differences in community composition are consistent.

Conclusions/Significance

Our data supports the notion that a divergent evolutionary scenario is driving community composition in the studied habitats. This work also improves the comprehension of microbial community dynamics in tropical waters and how they are structured in relation to physicochemical parameters. Furthermore, this paper reveals for the first time the pristine bacterioplankton communities in a tropical island at the South Atlantic Ocean.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Annual dynamics of bacterioplankton assemblages in the Gulf of Trieste (Northern Adriatic Sea)

Bacterioplankton community diversity was investigated monthly in coastal waters of the Gulf of Trieste (NE Adriatic Sea) throughout 2003. Superficial bacterial assemblages of two differently freshwater influenced stations were studied using PCR-DGGE fingerprinting techniques. Bacterial genetic diversity of the sampled area, as estimates of the number of DGGE bands was high (36-64) compared to that reported in other studies employing this fingerprint technique. The similarity index (Sorensen Index) between assemblages showed a defined operational taxonomic units (OTUs) succession pattern in the more typically marine station with stable winter communities and quickly changing summer ones. On the contrary in the station affected by riverine inputs no clear pattern was detected. In both sites, according to cluster analyses performed on the DGGE banding pattern, three seasonal assemblages were identified: winter-spring, summer and fall. Sequence analysis of fifty-six among the brightest gel bands led to the observation of bacteria affiliated to Gram positive, Cyanobacteria, Cytophaga-Flavobacteria-Bacteroides (CFB) lineages and the alpha-, gamma- and delta- subdivisions of the Proteobacteria. Gamma-Proteobacteria constituted the main fraction (60%) of sequences in the more typically marine station, whereas the river-influenced station was characterised by more heterogeneous assemblages (39% alpha-Proteobacteria, 32% Flavobacteria).     

Bacterial Community Succession in Natural River Biofilm Assemblages

Temporal bacterial community changes in river biofilms were studied using 16S rRNA gene-based polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) followed by sequence analysis. Naturally occurring biofilms were sampled in 2001 during an undisturbed 7-month low-water period in the River Garonne (SW France). During the sampling period epilithic biomass exhibited a particular pattern: two 3-month periods of accumulation that resulted in two peaks in summer and fall, each at about 25 g ash-free dry mass per square meter. Bacterial community DGGE profiles differed between the summer and fall biomass peaks and shared only 30% common operational taxonomic units (OTUs), suggesting the influence of seasonal factors on these communities. During the second biomass accrual phase, bacterial richness and the appearance of new OTUs fitted a conceptual model of bacterial biofilm succession. During succession, five OTUs (corresponding to Dechloromonas sp., Nitrospira sp., and three different Spirosoma spp.) exhibited particular patterns and were present only during clearly defined successional stages, suggesting differences in life-history strategies for epilithic bacteria. Co-inertia analysis of DGGE banding patterns and physical–chemical data showed a significant relationship between community structure and environmental conditions suggesting that bacterial communities were mainly influenced by seasonal changes (temperature, light) and hydrodynamic stability. Within the periods of stability, analysis of environmental variables and community patterns showed the dominant influence of time and maturation on bacterial community structure. Thus, succession in these naturally occurring epilithic biofilm assemblages appears to occur through a combination of allogenic (seasonal) and autogenic changes.     

Allochthonous inputs of riverine picocyanobacteria to coastal waters in the Arctic Ocean

The observed onset of climate change at high northern latitudes has highlighted the need to establish current baseline conditions in the Arctic Ocean, and has raised concern about the potential for the invasion and growth of biota that have warm temperature optima, such as cyanobacteria. In this study, we used 16S rRNA gene sequences as a molecular marker to evaluate the hypothesis that Arctic rivers provide a major inoculum of cyanobacteria into the coastal Arctic Ocean. Surface samples were collected along a transect extending from the Mackenzie River (Northwest Territories, Canada), across its estuary, to 200 km offshore at the edge of the perennial Arctic pack ice (Beaufort Sea). The highest picocyanobacteria concentrations occurred in the river, with concentrations an order of magnitude lower at offshore marine stations. The 16S rRNA gene clone libraries of five surface samples and five strains along this gradient showed that the cyanobacterial sequences were divided into eight operational taxonomic units (OTUs), six OTUs closely related to freshwater and brackish Synechococcus and two OTUs of filamentous cyanobacteria. No typically marine Synechococcus sequences and no Prochlorococcus sequences were recovered. These results are consistent with the hypothesis of an allochthonous origin of picocyanobacteria in the coastal Arctic Ocean, and imply survival but little net growth of picocyanobacteria under the present conditions in northern high-latitude seas.     

Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.     

Recombination and microdiversity in coastal marine cyanophages

Genetic exchange is an important process in bacteriophage evolution. Here, we examine the role of homologous recombination in the divergence of closely related cyanophage isolates from natural marine populations. Four core-viral genes (coliphage T4 homologues g20 , g23 , g43 and a putative tail fibre gene) and four viral-encoded bacterial-derived genes ( psbA , psbD , cobS and phoH ) were analysed for 60 cyanophage isolates belonging to five Rhode Island Myovirus (RIM) strains. Phylogenetic analysis of the 60 concatenated sequences revealed well-resolved sequence clusters corresponding to the RIM strain designations. Viral isolates within a strain shared an average nucleotide identity of 99.3–99.8%. Nevertheless, extensive microdiversity was observed within each cyanophage strain; only three of the 60 isolates shared the same nucleotide haplotype. Microdiversity was generated by point mutations, homologous recombination within a strain, and intragenic recombination between RIM strains. Intragenic recombination events between distinct RIM strains were detected most often in host-derived photosystem II psbA and psbD genes, but were also identified in some major capsid protein g23 genes. Within a strain, more variability was observed at the psbA locus than at any of the other seven loci. Although most of the microdiversity within a strain was neutral, some amino acid substitutions were identified, and thus microdiversity within strains has the potential to influence the population dynamics of viral–host interactions.     

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and β-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, β-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species. Received: 22 January 1999 / Accepted: 28 April 1999