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

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 per 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.

Effects of bacterial communities on biofuel-producing microalgae: stimulation, inhibition and harvesting

“…Bacterial communities can be monitored with appropriate molecular techniques, with the option of using real-time monitoring by real-time quantitative polymerase chain reaction (qPCR). This technique has been applied in many different samples to monitor shifts in bacterial communities, including human feces (Bartosch et al, 2004), deep sea sponges (Cassler et al, 2008), high-strength organic wastewater (Lee et al, 2008), soil (Fierer et al, 2005) and other environments (Shannon et al, 2007;Wang et al, 2010a;Yergeau et al, 2009). By applying this technique, the bacterial community associated with biofuel-producing microalgae could be monitored in real-time; ''desirable'' and ''deleterious'' bacterial symbionts could then be subsequently regulated.…”

Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

“…Other culture-independent studies employed PCR amplification of bacterial 16S rRNA genes directly from sponge metagenomic DNA followed by denaturing gradient gel electrophoresis [22,48] or restriction fragment length polymorphism analyses [23,61]. Cloning and sequencing of 16S rRNA genes has also been used in many microbial diversity investigations from a wide range of sponge species [5,11,15,27,35,42,[51][52][53]. Recently, pyrosequencing of PCR amplicon libraries from metagenomic sources has allowed for deeper insights into environmental microbial community structures, negating the requirement for a cloning step and providing numbers of sequencing reads orders of magnitude greater than was previously possible.…”

Quantitative comparison of bacterial communities in two Mediterranean sponges

“…The recent development of quantitative real time polymerase chain reaction (qPCR) has emerged as a practical method to quantify microorganisms in multiple environments (Skovhus et al 2004;Fierer et al 2005;Galand et al 2009). Despite the increasing interest in sponge symbiosis, the use of qPCR to quantify microbial symbionts in sponges has been reported only once (Cassler et al 2007).…”