Combined Microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ

We propose a novel method for studying the function of specific microbial g roups in situ. Since natural microbial communities are dynamic both in comp osition and in activities, we argue that the microbial "black box" should n ot be regarded as homogeneous. Our technique breaks down this black box wit h group-specific fluorescent 16S rRNA probes and simultaneously determines H-3-substrate uptake by each of the subgroups present via microautoradiogra phy (MAR). Total direct counting, fluorescent in situ hybridization, and MA R are combined on a single slide to determine (i) the percentages of differ ent subgroups in a community, (ii) the percentage of total cells in a commu nity that take up a radioactively labeled substance, and (iii) the distribu tion of uptake within each subgroup. The method was verified with pure cult ures. In addition, in situ uptake by members of the alpha subdivision of th e class Proteobacteria (alpha-Proteobacteria) and of the Cytophaga-Flavobac terium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these o rganisms account for a large portion of the picoplankton community in the s ample examined (similar to 60% of the universal probe-labeled cells and sim ilar to 50% of the total direct counts), but they also are significant in t he uptake of dissolved amino acids in situ. Nearly 90% of the total cells a nd 80% of the cells belonging to the alpha-Proteobacteria and Cytophaga-Fla vobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-trac king autoradiographic fluorescent in situ hybridization (STARFISH), which s hould aid in the "dissection" of microbial communities by type and function .