Single-cell RNA content of natural marine planktonic bacteria measured by hybridization with multiple 16S rRNA-targeted fluorescent probes

Despite the potential of cellular RNA as a tool for the study of bacterial activities in natural environments, cellular RNA has rarely been measured because of methodological limitations. In a previous study, we quantitatively analyzed single-cell RNA content with multiple 16S rRNA-targeted fluorescent probes. In the present study, we explore the potential of this approach with 12 samples of natural planktonic bacteria collcctcd from coastal water over a 6-month period (late winter to summer). We measured fluorescence from single cells hybridized with multiple probes, cellular DNA and RNA by ethidium bromide (EtBr) lluoromctry, and t3H]thymidine (TdR) incorporation rates. Fluorescence of the probehybridized cells was highly correlated to cellular RNA determined by EtBr fluorometry. Winter and summer samples showed different patterns of fluorescence-frequency distribution. Temperature had a large positive effect on TdR incorporation rates, but there was a negative correlation with cellular RNA content; only the samples taken at elevated summer temperatures showed a positive relationship between TdR incornoration and cellular RNA content. We estimated that the RNA content of the cells not visibly labeled wiih five probes was ~0.3 fg. The role of bacterioplankton in the aquatic food web has been examined extensively during the past decade. With the aid of new techniques, microbial biomass and activities have been m-examined, and new findings led to an increasing recognition of the importance of the microorganisms (Azam et al. 1983; Cho and Azam 1988; Fuhrman and Azam 1982). However, most current methods invariably measure bacterial activities averaged over the whole community (e.g. Fuhrman and Azam 1982). Despite the emerging evidence that natural bacterial communities contain a variety of different organisms (Giovannoni et al. 1990; Schmidt et al. 199 1; Ward et al. 1990) and that the species composition varies over time and space (Lee and Fuhrman 199 la), current methods do not provide information regarding the distribution of activity among different members of a community. ’ Corresponding author. 2 Current address: Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul, 425-600. Acknowledgments WC thank Edward DeLong for providing helpful information, Julie LaRoche for discussions and critical review, Jeneen Wagner for help with lab work, and anonymous reviewers for criticism. This work was supported by the U.S. Department of Energy under contract DE-AC02-76CHOOO 16. Many studies have shown that RNA content reflects the activities (e.g. growth) of diverse organisms (Bamstedt and Skjoldal 1980; Buckley 1984; Dortch et al. 1983; Kerkhof and Ward 1993). High correlations have been found between the growth rates of cultured, fastgrowing bacteria and their cellular RNA contents measured from nucleic acid preparations (Brcmer and Dennis 1987; DeLong et al. 1989; Schaechtcr et al. 1958; Kemp et al. 1993). The correlation results from the direct role of ribosomes in protein synthesis. However, the potential usefulness of cellular RNA content has been outweighed by methodological limitations or deficiencies. For example, the level of bacterial cellular RNA is very low and difficult to measure, and conventional methods that measure nucleic acids via extraction are unable to quantify RNA content on a singlecell basis. In addition, RNA degrades so easily that extra care must be taken when handling RNA samples, which makes the conventional methods impractical for routine use. A recently developed technique uses fluorescently labeled, 16S ribosomal RNA (rRNA)targeted oligonucleotide probes to identify individual cells by fluorescence microscopy (DeLong et al. 1989). The fluorescent-probe method provides a powerful tool to study the phylogeny of naturally occurring bacterial cells (Amann et al. 1990b; DeLong et al. 1989; Dis-