Ribosomal RNA content in microcolony forming soil bacteria measured by quantitative 16S rRNA hybridization and image analysis

The correlation between the growth rate, rRNA concentration and number of rrna operons was studied in Alcaligenes sp. A2, Pseudomonas fluorescens R2f and Bacillus sp. B1 cells grown exponentially in liquid 1/10 strength tryptic soy broth (1/10 TSB) medium and grown to microcolony (mCFU) size on polycarbonate membrane filters floating on 1/10 TSB. The rRNA concentration was also determined in cells forming mCFUs after incubation on membranes of rhizosphere and bulk soil samples taken from a barley (Hordeum vulgare) microcosm experiment. A protocol for fluorescent in situ hybridization directly on polycarbonate membrane filters combined with confocal laser scanning microscopy and image analysis was used to measure the probing intensity from individual cells within the mCFUs. We found that cells forming mCFU on membranes had a rRNA concentration slightly higher than cells grown in fluid medium. Mean values of growth rates and rRNA concentration measured as a probing intensity per cell area were 0.31, 0.61 and 0.63 h(-1) and 52, 63 and 146 units per cell area for Alcaligenes sp. A2, P. fluorescens R2f, and Bacillus sp. B1, respectively, when grown on membranes. The number of rrna operons carried by the three bacteria was determined to be between two to three, four and seven to nine. Alcaligenes sp. A2 which carried only two to three rrna operons had the lowest concentration of rRNA. Furthermore this organism had a lower growth rate compared to P. fluorescens R2f, and Bacillus sp. B1. Although P. fluorescens R2f and Bacillus sp. B1 had the same growth rate, Bacillus sp. B1 expressed a much higher rRNA concentration, which reflected well the high number of rrna operons carried by this bacterium. The mean probing intensity of mCFUs obtained after incubation of rhizosphere and bulk soil samples varied more than four-fold in both environments. The percentage of mCFU forming bacteria expressing a low concentration of rRNA was at least as high in the rhizosphere as in the bulk soil. The rationale behind carrying a high copy number of rrna operons and maintaining high concentrations of rRNA during growth is discussed.