Culture isolation and culture-independent clone libraries reveal new marine Synechococcus ecotypes with distinctive light and N physiologies

Abstract

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.

FIG. 1.

Hydrographic and cell abundance data for the water column from which samples were taken for the construction of clone libraries. This water column was typical of the Sargasso Sea at the time of sampling. Data for temperature (dotted line), relative chlorophyll fluorescence (solid line), Synechococcus cells (filled circles), Prochlorococcus cells (open circles), and picoeukaryotic phytoplankton (open diamonds) are shown.

FIG. 2.

Rarefaction curves of cyanobacterial phylotype diversity found for clone libraries constructed from depths of 5 m (A), 75 m (B), and 175 m (C). Error bars represent standard deviations of 100 replicate curves.

FIG. 3.

Phylogenetic tree of the genus marine Synechococcus inferred using 406 positions of the 16S-23S ITS region. The tree shown was constructed using the HKY85 model of nucleotide substitution and minimum evolution as the objective, but tree topologies found using other methods were similar (see Materials and Methods). Bootstrap values greater than 50 are given at the nodes and were determined using the HKY85 model with minimum evolution (1,000 replicates), the TrN+G model with minimum evolution (1,000 replicates), and the TrN+G model with maximum likelihood (100 replicates). Sequences from environmental clones are in italics. Sequences obtained in this study are in boldface type. The total numbers of sequences recovered for each clade are listed after the clade number designation (isolates obtained, sequences cloned).

FIG. 4.

N utilization experiments for clade XV strains UW69 (A) and UW106 (B) and clade II strain UW86 (C). Cultures were initially acclimated in 100 μM ammonium medium and transferred at time zero into medium containing no N (white diamonds) or 100 μM ammonium (black circles), nitrite (open squares), or nitrate (gray triangles). Symbols represent averages of triplicate tubes, and error bars are standard deviations of triplicates.

FIG. 5.

N utilization recovery experiments for clade XV strains UW69 (A) and UW106 (B). Cultures were initially acclimated in ASW with 100 μM ammonium and transferred at time zero into medium with 100 μM nitrate. Fluorescence units for the initial days are not shown because values were below the detection limit of the fluorometer. After 4 or 5 days (indicated by arrows), cultures were split into three treatments: 100 μM ammonium added (black circles), 100 μM nitrite added (open squares), and no additional N added (gray triangles). Symbols represent averages of triplicate tubes, and error bars are standard deviations of triplicates.

FIG. 6.

Phylogenetic relationships of marine Synechococcus strains inferred using narB sequences. The distance-based tree shown here was inferred using 630 nucleotides with the HKY+G substitution model and with minimum evolution as the criterion (see Materials and Methods). The best inferred tree using the 210 encoded amino acids and distances computed with the Dayhoff PAM matrix had a similar topology (data not shown). Bootstrap values greater than 50 are shown for 1,000 and 100 replicates of nucleotide- or amino acid-based trees, respectively.

FIG. 7.

Ratios of accessory pigments PUB and PEB determined by spectrofluorometry of cultures grown under white (white bars) and blue (black bars) constant light. Bars represent averages of two to four replicate cultures, and error bars represent standard deviations of those replicates. The clade numbers to which the strains belong are given under the strain names.

FIG. 8.

Absorbance spectra of in vivo cells (thick lines) or sonicated cell lysates (thin lines) for clade II strains UW86 and UW90 grown under constant white light. UW86 exhibits a peak at 545 nm, which is typical for marine Synechococcus strains and represents the accessory pigment PEB. Note that this peak is seen for strain UW90 lysate, but it is shifted to 570 nm in the spectrum of in vivo cells (arrow).