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. 2005 Nov;71(11):7019-28.
doi: 10.1128/AEM.71.11.7019-7028.2005.

Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora

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Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora

Tracy J Mincer et al. Appl Environ Microbiol. 2005 Nov.

Abstract

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.

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Figures

FIG.1.
FIG.1.
Predicted secondary structures of Salinispora 16S rRNA. (A) Detail of the variable loop containing Salinispora-specific signature nucleotides (U, U) at positions 442 to 443 (467 to 468 by E. coli numbering) used to design the F468 primer. (B) Consensus secondary structure of S. arenicola strain CNH-721 (GenBank accession number AY878316) drawn using Streptomyces coelicolor as a reference (5). Helix loop structures in hypervariable regions were confirmed by using the Mfold program (29). Genus (*)- and species (#)-specific signatures are identified. Position 1002 is the site of the C→T transition observed in the uncultured S. arenicola phylotype III.
FIG. 2.
FIG. 2.
Ethidium bromide-stained 3% agarose gel comparing HaeIII restriction endonuclease digests of Salinispora seminested PCR amplification products. Lanes: M, 100-bp marker; 1, S. tropica cultivated isolate; 2, S. arenicola cultivated isolate; 3, uncultivated phylotype III from sediment BA02-36 environmental library; 4, sample BA02-35 enrichment (sediment extract and rifampin) showing the S. tropica RFLP pattern; 5, sample BA02-35 enrichment (sediment extract and novobiocin) showing the S. tropica RFLP pattern; 6, sample BA02-36 enrichment (sediment extract and rifampin) showing the predominant S. arenicola RFLP pattern; 7, sample BA02-36 enrichment (sediment extract and novobiocin) showing the S. arenicola RFLP pattern; 8, sample BA03-03 enrichment (sediment extract and rifampin) showing the S. tropica RFLP pattern; 9, sample BA03-03 enrichment (sediment extract and novobiocin) showing the S. tropica RFLP pattern.
FIG. 3.
FIG. 3.
(A) Neighbor-joining phylogenetic dendrogram constructed with nearly full 16S rRNA gene sequences (1,407 unambiguous nucleotides) illustrating cultured Salinispora species diversity including strains isolated from the Red Sea (CNH-725), the Sea of Cortez (CNH-964), Palau (CNS-143), and Guam (CNR-425) in comparison to all formally described genera within the Micromonosporaceae (List of Bacterial Names with Standing in Nomenclature [http://www.bacterio.cict.fr/]) for which sequence data was available from the NCBI Web site. Strain CNS-143 was cultured as part of a separate study and is currently being examined to determine whether it represents a third Salinispora species. Genera other than Salinispora that contain multiple species are represented with a triangle with the number of species included in parentheses. Propionibacterium propionicus, Streptosporangium corrugatum, and Streptomyces lividans were used as outgroups. In both panels A and B, bootstrap values were calculated from 1,000 resamplings and are shown at their respective nodes for values ≥60%. (B) Neighbor-joining phylogenetic dendrogram, obtained after seminested PCR amplification of the 16S rRNA gene, using 995 unambiguous nucleotides illustrating cultured and culture-independent Salinispora diversity. Clones were derived from deep (BA02-36) and shallow (BA00-11) samples with the number of identical clones and accession numbers noted in parentheses. Strain CNS-143 was isolated from Palau and may represent a new species (subject of a separate study). Micromonospora olivasterospora and Dactylosporangium aurantiacum (not shown) were used as outgroups.

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