Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales

Abstract

A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591-599, 1996) yielded four clones with 16S rRNA genes from the order Planctomycetales. Three of the clones belong to the Pirellula group and one clone belongs to the Planctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

FIG. 1

Southern blot of EcoRI- and HindIII-digested fosmids hybridized with ³²P end-labeled Planctomycetales-specific 16S rRNA and universal rRNA oligonucleotides. Sizes (in base pairs) are indicated on the left. Lanes: 1, 5H12; 2, 6O13; 3, 6N14; 4, 7F15.

FIG. 2

Evolutionary relationships among Planctomycetales 16S rRNA genes found in cultured isolates, environmental clone libraries generated by PCR, and fosmid clones. Sequences were aligned by eye. Phylogenetic analyses included all nucleotide positions for which unambiguously determined and aligned sequence data were available for the strains and clones examined. Since sequences in public databases are sometimes shorter than the ca. 1,550 bases determined for our fosmid clones, phylogenetic analyses included fewer nucleotide positions. Phylogenetic frameworks were inferred by neighbor-joining with Kimura 2-parameter genetic distances (2:1 transition:transversion ratio) with the PHYLIP version 3.5c computer program (5a). Bootstrap proportions were calculated from 100 resampled data sets by both neighbor-joining and maximum parsimony algorithms. Bootstrap proportions are indicated by small numbers; neighbor-joining proportions are shown above, and maximum parsimony proportions are shown below. Scale bars indicate the inferred frequency of substitutions per nucleotide position. Strains labeled “Schlesner” are from reference . (a) Pirellula group phylogeny inferred from 226 nucleotide positions. (b) Planctomyces group phylogeny inferred from 321 positions.