Phylum- and class-specific PCR primers were tested for the production of clone libraries and for denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Primers were ...designed to specifically amplify 16S rRNA gene fragments of the phyla Bacteroidetes, Planctomycetes and Firmicutes, of three classes of the phylum Proteobacteria, the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, and of the Cyanobacteria (including chloroplast 16S rRNA genes). The specificity of the seven primer pairs was tested by producing clone libraries from environmental DNA samples from mesotrophic (Norwegian coastal) and oligotrophic (Northern Atlantic Gyre) environments. Five of the seven primer pairs specifically amplified target 16S rRNA gene sequences. Exceptions were the Betaproteobacteria- and Firmicutes-specific primers, which were relatively successful with coastal water mesocosm samples but less so with the Northern Atlantic Gyre sample. Phylogenetic analysis of sequences from the Gammaproteobacteria clone library revealed that the coastal sample yielded a number of clones that clustered within clades that belong to the oligotrophic marine Gammaproteobacteria (OMG) group, indicating that this group is not confined exclusively to the oligotrophic environment. Comparison of the bacterial diversity of the environmental DNA sample from the coastal and the open ocean using a two- or three-step nested PCR-DGGE process revealed significant differences in the bacterial communities. The application of the group-specific primers provides a higher resolution genetic fingerprinting approach than existing DGGE primer sets.
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To mark the 200
th
anniversary of Charles Darwin's birth, a special event was held at Oxford, which included a 'Conversation' between Professor Richard Dawkins and Bishop Richard Harries. Here we ...present a personal reminiscence of the event.
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Based on the increase of 16S rRNA gene sequences in databases it is possible to design improved oligonucleotide primers for this gene. Primers were designed in silico to specifically amplify ...fragments of the gene from the Alpha, Beta and Gamma subgroups of the Proteobacteria, as well as from Bacteroidetes, Firmicutes, Cyanobacteria and Planctomycetes and tested in silico and in vitro. The aim was to investigate bacterioplankton diversity and reveal greater fingerprint diversity within these groups than is possible using primers specific for the entire domain Bacteria, and also to reduce clone library redundancy. It was then aimed to investigate the potential impacts of increased pCO2 and ocean fertilisation with iron (Fe) and phosphorus (P), on bacterioplankton diversity. Group-specific clone libraries representing contrasting marine regions were analysed, and the usefulness and specificity of the primers validated. The clone libraries showed members of the oligotrophic marine group (OMG) to be present in an in situ coastal mesocosm supplemented with nutrients. The newly-developed group-specific primers were used in combination with an improved method of denaturing gradient gel electrophoresis (DGGE) to profile in detail bacterial communities in mesocosms, which were maintained at 750 ppm of pCO2, the level projected for the global surface ocean in the year 3000, and 380 ppm of CO2, the present level. Increased pCO2 correlated with a decrease in abundance of some members of the Gammaproteobacteria. Otherwise there was little impact on diversity due to raised pCO2. The same DGGE protocol was applied to samples from an ocean Fe and P fertilisation experiment. Diversity change due to Fe was not evident. However in seawater amended with P there was an explosive growth of some cells with 16S rRNA genes similar to those of the SAR86 clade, and others with similarity to Gammaproteobacteria with large genomes such as Oceanospirillum sp. and Psychromonas sp.
Based on the increase of 16S rRNA gene sequences in databases it is possible to design improved oligonucleotide primers for this gene. Primers were designed in silico to specifically amplify ...fragments of the gene from the Alpha, Beta and Gamma subgroups of the Proteobacteria, as well as from Bacteroidetes, Firmicutes, Cyanobacteria and Planctomycetes and tested in silico and in vitro. The aim was to investigate bacterioplankton diversity and reveal greater fingerprint diversity within these groups than is possible using primers specific for the entire domain Bacteria, and also to reduce clone library redundancy. It was then aimed to investigate the potential impacts of increased pCO2 and ocean fertilisation with iron (Fe) and phosphorus (P), on bacterioplankton diversity. Group-specific clone libraries representing contrasting marine regions were analysed, and the usefulness and specificity of the primers validated. The clone libraries showed members of the oligotrophic marine group (OMG) to be present in an in situ coastal mesocosm supplemented with nutrients. The newly-developed group-specific primers were used in combination with an improved method of denaturing gradient gel electrophoresis (DGGE) to profile in detail bacterial communities in mesocosms, which were maintained at 750 ppm of pCO2, the level projected for the global surface ocean in the year 3000, and 380 ppm of CO2, the present level. Increased pCO2 correlated with a decrease in abundance of some members of the Gammaproteobacteria. Otherwise there was little impact on diversity due to raised pCO2. The same DGGE protocol was applied to samples from an ocean Fe and P fertilisation experiment. Diversity change due to Fe was not evident. However in seawater amended with P there was an explosive growth of some cells with 16S rRNA genes similar to those of the SAR86 clade, and others with similarity to Gammaproteobacteria with large genomes such as Oceanospirillum sp. and Psychromonas sp.
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