Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean

Abstract

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.

FIG. 1

The Columbia River estuary with sampling sites.

FIG. 2

Phylogenetic relationships among 16S rRNA sequences from Columbia River, estuary, and adjacent coastal ocean clones and from other environmental clones and cultured organisms. (A) α-proteobacteria; (B) β-proteobacteria; (C) γ-proteobacteria; (D) δ-proteobacteria; (E) Verrucomicrobiales and Planctomyces clade; (F) Cytophaga-Flexibacter assemblage; (G) gram-positive bacteria; (H) chloroplasts and cyanobacteria; (I) all other clones. Fifty percent majority-rule trees were constructed by the neighbor-joining method. The percentages of 1,000 bootstrap replicates that supported the branching order are shown above or near the relevant nodes. The scale bars correspond to a 10% difference in nucleotide sequence. Clones from this study are indicated in boldface and are named with the following prefixes, designating their sources: CR, Columbia River; CRE, Columbia River estuary; CRO, coastal ocean; PA, particle attached; and FL, free-living. All sequences are available from the GenBank database, and accession numbers are provided if the organism or clone name is not unique. CFB, Cytophaga-Flexibacter-Bacteroides phylum.

FIG. 2

Phylogenetic relationships among 16S rRNA sequences from Columbia River, estuary, and adjacent coastal ocean clones and from other environmental clones and cultured organisms. (A) α-proteobacteria; (B) β-proteobacteria; (C) γ-proteobacteria; (D) δ-proteobacteria; (E) Verrucomicrobiales and Planctomyces clade; (F) Cytophaga-Flexibacter assemblage; (G) gram-positive bacteria; (H) chloroplasts and cyanobacteria; (I) all other clones. Fifty percent majority-rule trees were constructed by the neighbor-joining method. The percentages of 1,000 bootstrap replicates that supported the branching order are shown above or near the relevant nodes. The scale bars correspond to a 10% difference in nucleotide sequence. Clones from this study are indicated in boldface and are named with the following prefixes, designating their sources: CR, Columbia River; CRE, Columbia River estuary; CRO, coastal ocean; PA, particle attached; and FL, free-living. All sequences are available from the GenBank database, and accession numbers are provided if the organism or clone name is not unique. CFB, Cytophaga-Flexibacter-Bacteroides phylum.

FIG. 2

Phylogenetic relationships among 16S rRNA sequences from Columbia River, estuary, and adjacent coastal ocean clones and from other environmental clones and cultured organisms. (A) α-proteobacteria; (B) β-proteobacteria; (C) γ-proteobacteria; (D) δ-proteobacteria; (E) Verrucomicrobiales and Planctomyces clade; (F) Cytophaga-Flexibacter assemblage; (G) gram-positive bacteria; (H) chloroplasts and cyanobacteria; (I) all other clones. Fifty percent majority-rule trees were constructed by the neighbor-joining method. The percentages of 1,000 bootstrap replicates that supported the branching order are shown above or near the relevant nodes. The scale bars correspond to a 10% difference in nucleotide sequence. Clones from this study are indicated in boldface and are named with the following prefixes, designating their sources: CR, Columbia River; CRE, Columbia River estuary; CRO, coastal ocean; PA, particle attached; and FL, free-living. All sequences are available from the GenBank database, and accession numbers are provided if the organism or clone name is not unique. CFB, Cytophaga-Flexibacter-Bacteroides phylum.

FIG. 2

Phylogenetic relationships among 16S rRNA sequences from Columbia River, estuary, and adjacent coastal ocean clones and from other environmental clones and cultured organisms. (A) α-proteobacteria; (B) β-proteobacteria; (C) γ-proteobacteria; (D) δ-proteobacteria; (E) Verrucomicrobiales and Planctomyces clade; (F) Cytophaga-Flexibacter assemblage; (G) gram-positive bacteria; (H) chloroplasts and cyanobacteria; (I) all other clones. Fifty percent majority-rule trees were constructed by the neighbor-joining method. The percentages of 1,000 bootstrap replicates that supported the branching order are shown above or near the relevant nodes. The scale bars correspond to a 10% difference in nucleotide sequence. Clones from this study are indicated in boldface and are named with the following prefixes, designating their sources: CR, Columbia River; CRE, Columbia River estuary; CRO, coastal ocean; PA, particle attached; and FL, free-living. All sequences are available from the GenBank database, and accession numbers are provided if the organism or clone name is not unique. CFB, Cytophaga-Flexibacter-Bacteroides phylum.

FIG. 2

Phylogenetic relationships among 16S rRNA sequences from Columbia River, estuary, and adjacent coastal ocean clones and from other environmental clones and cultured organisms. (A) α-proteobacteria; (B) β-proteobacteria; (C) γ-proteobacteria; (D) δ-proteobacteria; (E) Verrucomicrobiales and Planctomyces clade; (F) Cytophaga-Flexibacter assemblage; (G) gram-positive bacteria; (H) chloroplasts and cyanobacteria; (I) all other clones. Fifty percent majority-rule trees were constructed by the neighbor-joining method. The percentages of 1,000 bootstrap replicates that supported the branching order are shown above or near the relevant nodes. The scale bars correspond to a 10% difference in nucleotide sequence. Clones from this study are indicated in boldface and are named with the following prefixes, designating their sources: CR, Columbia River; CRE, Columbia River estuary; CRO, coastal ocean; PA, particle attached; and FL, free-living. All sequences are available from the GenBank database, and accession numbers are provided if the organism or clone name is not unique. CFB, Cytophaga-Flexibacter-Bacteroides phylum.

FIG. 3

(A) Longitudinal cross section of an ETM region of the Columbia River estuary, showing inputs of river and coastal ocean water and particles and the location of the ETM at the head of the salt wedge. Curved arrows indicate mixing of freshwater (white) and seawater (dark gray). (B) Compositions of clone libraries at the phylum and subphylum levels. Arrows show movement of bacterial types from source populations into the estuary. Estuarine clone libraries are separated into clones unique to the estuary (bottom section of free-living and particle-attached charts), clones similar to those found in the river (upper left sections), and clones similar to those found in the coastal ocean (upper right sections). Estuarine clones were designated river or coastal ocean when they clustered with clones from these source communities. Most had at least 96% sequence similarity to river and coastal ocean clones.