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. 2003 May;69(5):2430-43.
doi: 10.1128/AEM.69.5.2430-2443.2003.

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Nicholas J Fuller  1 , Dominique Marie, Frédéric Partensky, Daniel Vaulot, Anton F Post, David J Scanlan
Affiliations

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Nicholas J Fuller et al. Appl Environ Microbiol. 2003 May.

Abstract

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.

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Figures

FIG. 1.
FIG. 1.
Agarose gel (A) and corresponding Southern blot (B) showing the two 16S rDNA copies from Synechococcus sp. strain WH 7803 cloned into pUC19, probed with digoxigenin-labeled 16S rRNA PCR product amplified from the same strain. Lanes: 1, strain WH 7803 genomic DNA; 2 and 3, pUC19 containing a 16S rDNA copy on a 4.0- and 4.6-kb HindIII fragment, respectively.
FIG. 2.
FIG. 2.
RFLP analysis of Synechococcus sp. isolates from the Red Sea. PCR amplicons of ntcA were digested with HaeIII and BstUI and separated by electrophoresis on a 10% polyacrylamide gel, revealing six different RFLP patterns. Patterns from other Synechococcus strains are shown for comparison. Lanes 1 to 14: Synechococcus sp. strains RS9901, RS9902, RS9903, RS9904, RS9905, RS9907, RS9908, RS9910, RS9911, RS9915, RS9916, RS9919, RS9920, and RS9921, respectively. Lanes 15 to 19: Synechococcus sp. strains WH 7803, WH 8018, WH 8103, Minos01, and CC9311, respectively.
FIG. 3.
FIG. 3.
Neighbor-joining phylogenetic tree of 16S rDNA sequences from marine Synechococcus and Prochlorococcus strains, with Jukes-Cantor correction. Partial sequences (<1,390 nucleotides) were added to the tree by using a maximum-parsimony option within ARB. The freshwater strain PCC6301 was used as the root. The tree was constructed with a filter of 1,345 nucleotides that excluded alignment positions with sequence ambiguity or missing data. The confidence of branch points was determined by three separate analyses (maximum likelihood, neighbor joining, and maximum parsimony), with multifurcations indicating branch points that were collapsed until supported in all three analyses by using a strict consensus rule. Bootstrap values (100 replicates) are shown from neighbor-joining analysis with Jukes-Cantor correction. Closed symbols represent values of >95%, open symbols represent values of 70 to 95%, and values of <70% are not shown. Circles represent values where full-length sequences (1,345 nucleotides) were available for the analysis; triangles represent values where shorter sequences (681 nucleotides) were used.
FIG. 4.
FIG. 4.
Neighbor-joining tree of 16S rDNA sequences from cyanobacterial strains, with Jukes-Cantor correction. The positions of Synechococcus MC-A, MC-B, and MC-C and their current phylogenetic assignments are indicated by brackets. *, strain WH 8101 was previously designated a member of MC-B (subcluster 5.2) but is designated here a member of Synechococcus subcluster 5.1. For Synechococcus sp. strain G2.1, only a 951-bp sequence was available and this was added to the tree by using a maximum-parsimony option within ARB. Escherichia coli was used as the root. The tree was constructed with a filter of 1,263 nucleotides that excluded alignment positions with sequence ambiguity or missing data. The confidence of branch points was determined by three separate analyses (maximum likelihood, neighbor joining, and maximum parsimony), with multifurcations indicating branch points that were collapsed until supported in all three analyses by using a majority consensus rule. Bootstrap values (100 replicates) are shown from neighbor-joining analysis with Jukes-Cantor correction. Closed symbols represent values of >95%, open symbols represent values of 70 to 95%, and values of <70% are not shown. Circles represent values where full-length sequences (1,263 nucleotides) were available for the analysis; triangles represent values where shorter sequences (877 nucleotides) were used.
FIG. 5.
FIG. 5.
Pie chart of the proportion of cultured isolates from the Red Sea belonging to particular Synechococcus clades.
FIG. 6.
FIG. 6.
Representative dot blots showing the specificity of hybridization of each Synechococcus clade-specific oligonucleotide (SYNn) to arrays of control 16S rDNA amplicons (Synechococcus sp. strain RS9903, Synechococcus 16S rDNA clone 5X15, Synechococcus sp. strains RS9913, RS9901, WH 8103, CC9311, WH 7803, WH 8018, RS9920, and Minos01 and Cyanobium sp. strain NS01). EUB338, eubacterial probe.
FIG. 7.
FIG. 7.
Ancillary CTD, flow cytometry, and dot blot hybridization data from a depth profile at station A, Gulf of Aqaba, Red Sea on 27 April 1999. (A) Temperature (· · · ·), chlorophyll a concentration (in micrograms liter−1) (+), and Synechococcus (▪), Prochlorococcus (•), and photosynthetic picoeukaryote (▴) cell numbers. (B) Relative hybridization abundance of Synechococcus clades detected by 16S rDNA oligonucleotide probes SYN1006RS (clade II) (▪), SYN1258 (clades I to VII and X) (•), and SYN262 (clade III) (▴), quantified from dot blots.
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