Title

Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp.

Authors

M. Cassler, MRI Florida Division
C. L. Peterson, Historic Bok Sanctuary
A. Ledger, Harbor Branch Oceanographic Institution, Inc
Shirley A. Pomponi, Harbor Branch Oceanographic Institution, Inc
A. E. Wright, Harbor Branch Oceanographic Institution, Inc
R. Winegar, MRI Florida Division
P. J. McCarthy, Harbor Branch Oceanographic Institution, Inc
Jose V. Lopez, Harbor Branch Oceanographic InstitutionFollow

Document Type

Article

Publication Date

4-2008

Publication Title

Microbial Ecology

Keywords

Bacteriology, Sponges (invertebrates), Polymerase chain reaction, Bacteria phylogeny

ISSN

0095-3628

Volume

55

Issue/No.

3

First Page

384

Last Page

394

Abstract

In this report, real-time quantitative PCR (TaqMan® qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan® qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan® assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies mer microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Comments

©Springer Science+Business Media, LLC 2007

NSUWorks Citation

Cassler, M.; C. L. Peterson; A. Ledger; Shirley A. Pomponi; A. E. Wright; R. Winegar; P. J. McCarthy; and Jose V. Lopez. 2008. "Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp.." Microbial Ecology 55, (3): 384-394. doi:10.1007/s00248-007-9283-5.

ORCID ID

0000-0002-1637-4125

ResearcherID

F-8809-2011

DOI

10.1007/s00248-007-9283-5