Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of ¹³ C-Substrate Incorporation by Microorganisms in Marine Sediments

Abstract

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13 C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13 C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13 C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13 C-labeled compounds. Similar results were obtained with the 13 C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13 C analysis of the captured SSU rRNA.

Assessing nutrient limitation of Prochlorococcus in the North Pacific subtropical gyre by using an RNA capture method

“…MacGregor et al (2002) demonstrated the ability of their hybridization capture method to isolate rRNA using biotinylated oligonucleotide probes and streptavidin-coated magnetic beads. Although MacGregor et al (2006) have subsequently updated their method, here we have applied the method as originally described (MacGregor et al 2002) with almost no changes, save for the addition of an internal recovery standard to make the method quantitative. Briefly, a hybridization mixture was created by mixing 10 ml of the 33 P-labeled sample RNA, 10 mL of 32 P-labeled standard RNA, 20 of mL formamide (discussed below), and 60 mL of hybridization buffer.…”

“…RIBOTRACE-We developed modifications to the RNA hybridization and capture method described by MacGregor et al (2002) to accommodate the use of radioactive internal recovery standards as described by Van Mooy et al (2004). We have termed this method RIBOTRACE.…”

“…Our new method is a hybrid of one method used to isolate oligonucleotide probe-defined RNA by hybridization and capture (MacGregor et al 2002), and another method used to quantify the incorporation of 33 P into primer-defined deoxyribonucleic acid (DNA) by hybridization and capture (Van Mooy et al 2004). This hybrid method, termed radioisotope-based tracking of ribonucleic acid synthesis by hybridization and capture (RIBOTRACE), was used to measure RNA synthesis by Prochlorococcus during two Hawaiian ocean time-series (HOT; Karl and Lukas 1996) cruises to Sta.…”

Detection and Capturing of 14C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads

“…4b). The use of the same archaeal and eukaryotic probes under the same hybridization conditions was previously shown to yield detectable hybridization products [10]. Therefore, the failure to detect the RNA of Archaea and eukaryotes in our mats indicates that they are either very low in abundance or they are not playing a role in the carbon cycling in these mats.…”

“…13 C or 15 N labeled) from natural samples by density-gradient centrifugation after incubation with isotope-labeled substrate [7,8], followed by sequencing and identification of key microorganisms involved in the assimilation of this substrate. The technique provided more sensitive results when performed on RNA or polar-lipid-derived fatty acids [9][10][11]. The isotope measurements in single microbial cells became possible with the help of isotopic measurement using high resolution ion microprobe, a technique known as NanoSIMS [12].…”