Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing

Abstract

The identification of the prokaryotic species which constitute marine bacterioplankton communities has been a long-standing problem in marine microbiology. To address this question, we used the polymerase chain reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by blunt-end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and hybridizations to oligonucleotide probes for the SAR11 and marine Synechococcus phylogenetic groups indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined completely. In addition to 16S rRNA genes from the marine Synechococcus cluster and the previously identified but uncultivated microbial group, the SAR11 cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field. Nature (London) 345:60-63], two new gene classes were observed. Phylogenetic comparisons indicated that these belonged to unknown species of alpha- and gamma-proteobacteria. The data confirm the earlier conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists.