Identification and targeted cultivation of abundant novel freshwater sphingomonads and analysis of their population substructure

Abstract

Little is known with respect to bacterial population structures in freshwater environments. Using complementary culture-based, cloning, and high-throughput Illumina sequencing approaches, we investigated microdiverse clusters of bacteria that comprise members with identical or very similar 16S rRNA gene sequences. Two 16S rRNA phylotypes could be recovered by cultivation in low-nutrient-strength liquid media from two lakes of different trophic status. Both phylotypes were found to be physiologically active in situ throughout most of the year, as indicated by the presence of their rRNA sequences in the samples. Analyses of internal transcribed spacer (ITS1) sequences revealed the presence of seven different sequence types among cultured representatives and the cloned rrn fragments. Illumina sequencing yielded 8,576 ITS1 sequences that encompassed 15 major and numerous rare sequence types. The major ITS1 types exhibited distinct temporal patterns, suggesting that the corresponding Sphingomonadaceae lineages occupy different ecological niches. However, since strains of the same ITS1 type showed highly variable substrate utilization patterns, the potential mechanism of niche separation in Sphingomonadaceae cannot be explained by substrate utilization alone and may be related to other traits.

Fig. 1.

Fractions of 16S rRNA gene sequences within the clone library of Walchensee bacterioplankton affiliated with the different families of Alphaproteobacteria.

Fig. 2.

(A) Maximum likelihood phylogenetic tree of almost-full-length Sphingomonadaceae 16S rRNA sequences obtained in the present study (in boldface). Eighty sequences of the environmental clone library, 117 sequences originating from primary liquid enrichment cultures, and 111 sequences of isolated pure cultures of Sphingomonadaceae were included in the analysis. The most abundant phylotypes, G1A and G7A, are indicated by gray boxes. Bar represents 0.01 fixed-point mutations per nucleotide. Values at nodes give bootstrap values in percentages (out of 1,000 bootstrap resamplings; only values of ≥50% are given). (B) Frequency of 16S rRNA sequence types from Walchensee lake present in the clone library (light gray columns) and enrichments (dark gray columns) (both from December 2007 samples) and among pure isolates from December 2007 (black columns) and August 2008 (hatched columns).

Fig. 3.

(A) Seasonal changes in the composition (based on the analysis of 16S rRNA genes; labeled DNA) and in the composition of the active fraction (based on the analysis of rRNA-cDNA; labeled cDNA) of Sphingomonadaceae in Walchensee and Starnberger See lakes. A negative image of a SybrGold-stained denaturing gradient gel is shown. Fragments were amplified using a PCR protocol specific for Sphingomonadaceae 16S rRNA genes. For comparison, the fingerprints of an isolated representative of phylotype G1A (isolate 505) and phylotype G7A (isolate 407), as well as fingerprints of cloned sequences of phylotypes G1A, G5B, and G7A (compare to Fig. 2A), are shown. (B) Cluster analysis of DGGE fingerprint patterns of seasonal Sphingomonadaceae communities using UPGMA. Values at nodes give bootstrap values in percentages (out of 10,000 bootstrap resamplings; only values of ≥50% are given). W, Walchensee lake; S, Starnberger See lake.

Fig. 4.

Cultivation success of planktonic bacteria (white columns, left ordinate) and percentage of Sphingomonadaceae cultures among the primary enrichments (black columns, right ordinate) derived from bacterioplankton communities in Walchensee and Starnberger See lakes. Cultivation efficiency is given as the percentage of total bacterial cell counts. Error bars indicate 95% confidence intervals.

Fig. 5.

Annual fluctuations of G1A ITS types in Walchensee (A) and Starnberger See (B) lakes. (C) Active fraction of G1A ITS1 types detected in cDNA samples. The remaining ITS types were present at a relative abundance of <5% and are not shown.

Fig. 6.

Variances of individual phenotypic traits. The box indicates the first and third quartile, the horizontal line in the box the median value, and the whiskers extend to a maximum of 1.5-fold of the box size. The values above the threshold value of the third quartile of ITS type 3 strains are depicted as open triangles for all data. The highly variable substances coded by the open triangles are listed in Table S4 in the supplemental material.