Diversity and seasonal variability of beta-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning

Abstract

The beta-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for beta-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described betaI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga: RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the beta-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.

FIG. 1.

Rooted phylogenetic consensus trees of 16S rRNA sequences showing affiliation of Elbe (Elb) and Spittelwasser (Spb) biofilm clones within the β-proteobacteria. The scale bar corresponds to a 10% difference in nucleotide sequence. E. coli was used as an outgroup (data not shown). The number of identical sequences (above 99% similarity) not shown in the tree is given in parenthesis (+1, etc.). Corresponding bands with the community pattern and accession numbers are indicated. Clamps mark river biofilm clusters (RBFs 1 to 9) containing at least two different biofilm clones, together with their most closely related environmental clones and cultured organisms (above 96% similarity). All sequences are available from the EMBL database. (A) Partial tree of the Comamonadaceae showing RBFs 1 to 3 within the cosmopolitan freshwater cluster βI (14). (B) Partial tree showing RBFs 4 to 7 in the Leptothrix subcluster of the β-proteobacteria as well as the affiliation of RBFs 8 and 9 and freshwater clusters βII to βIV (14).

FIG. 1.

Rooted phylogenetic consensus trees of 16S rRNA sequences showing affiliation of Elbe (Elb) and Spittelwasser (Spb) biofilm clones within the β-proteobacteria. The scale bar corresponds to a 10% difference in nucleotide sequence. E. coli was used as an outgroup (data not shown). The number of identical sequences (above 99% similarity) not shown in the tree is given in parenthesis (+1, etc.). Corresponding bands with the community pattern and accession numbers are indicated. Clamps mark river biofilm clusters (RBFs 1 to 9) containing at least two different biofilm clones, together with their most closely related environmental clones and cultured organisms (above 96% similarity). All sequences are available from the EMBL database. (A) Partial tree of the Comamonadaceae showing RBFs 1 to 3 within the cosmopolitan freshwater cluster βI (14). (B) Partial tree showing RBFs 4 to 7 in the Leptothrix subcluster of the β-proteobacteria as well as the affiliation of RBFs 8 and 9 and freshwater clusters βII to βIV (14).

FIG. 2.

Community profiles of β-proteobacteria based on specific amplification and TGGE separation of 16S rRNA gene sequences (positions 8 to 381) from 1-month-old biofilms grown in the Elbe and Spittelwasser Rivers. Numbers indicate bands for which corresponding clones were found, and letters mark bands for which no corresponding clones were found. EΣ and SΣ are abbreviations for river-specific references (ΣElbe and ΣSpittelwasser) generated from pooled PCR products from all sampling dates. The month of sampling is indicated above the lanes (Jun, June; Jul, July; Aug, August; Sep, September; Nov, November; Dec, December; Jan, January; Feb, February; Mar, March). Hyphens between river-specific references mark bands which were present in both rivers.