Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria

Abstract

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.

FIG. 1.

Maximum-likelihood phylogenetic tree constructed using partial 16S rRNA gene sequences of closely related speciated and nonspeciated strains belonging to the alpha- and gamma-Proteobacteria. The tree is rooted using Nitrococcus mobilis (L35510) as the outgroup. Nucleotide sequence database accession codes are shown in the brackets. The length of each branch is proportional to the estimated number of substitutions per position. Sequenced strains are highlighted in boxes; most-likely identities were assigned.

FIG. 2.

. Maximum-likelihood phylogenetic tree constructed using partial 16S rRNA gene sequences of closely related speciated and nonspeciated strains belonging to the high-G+C gram-positive bacteria. The tree is rooted using Acidimicrobium ferrooxidans (U75647) as the outgroup. Nucleotide sequence database accession codes are shown in the brackets. The length of each branch is proportional to the estimated number of substitutions per position. Sequenced strains are highlighted in boxes; most-likely identities were assigned.

FIG. 3.

Visual coaggregation scores for different pairs of strains. Cells were grown to stationary phase and harvested after 36, 71, or 144 h. Sphingomonas sp. strain 2.10 and Afipia sp. strain 2.2 (36 h) showed a score of 0 (no coaggregation), P. marcusii 2.21 and B. natatoria 2.3 (72 h) showed a score of 1+, Sphingomonas sp. strain 2.18 and Afipia sp. strain 2.2 (144 h) showed a score of 2+, B. natatoria 2.1 and Nocardioides sp. strain 2.20 (144 h) showed a score of 3+, and B. natatoria 2.1 with M. luteus 2.13 (72 h) showed a score of 4+. Bar, 1 cm.

FIG. 4.

Light micrographs showing rod-shaped cells of B. natatoria 2.1 coaggregating to tetrads of coccus-shaped cells of M. luteus 2.13. Bar, 10 μm.

FIG. 5.

A diagrammatic representation of the specificity of coaggregation of all 19 identified strains when grown separately in batch culture and harvested after 36, 72, and 144 h. Optimal expression of coaggregation at either 36 (—), 72 (- - -), or 144 h is shown for each pair. Expression of coaggregation that was constant between pairs at 36, 72, and 144 h is also shown (-^..-^..-). Visual coaggregation scores below a score of 2 are not included.