Application of a strain-specific rRNA oligonucleotide probe targeting Pseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment

Abstract

Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain. The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P. fluorescens Ag1. The correlation between the ribosomal content of P. fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h. Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content. The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy. After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell. After 1 day of carbon starvation, the rRNA content had decreased to 20%. When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30 degrees C. P. fluorescens Ag1 was used in a mesocosm release experiment. The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces. The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P. fluorescens Ag1 in the mesocosm experiment.