Quantification of bacterial mRNA involved in degradation of 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 from liquid culture and from river sediment by reverse transcriptase PCR (RT/PCR).

ABSTRACT

Competitive reverse transcriptase polymerase chain reaction (RT/PCR) was used to quantify the mRNA of the tcbC gene of Pseudomonas sp. strain P51. The tcbC gene encodes the enzyme chlorocatechol-1,2-dioxygenase involved in 1,2,4-trichlorobenzene (TCB) degradation. The mRNA content per cell was monitored in a batch culture growing on 1,2,4-TCB. No mRNA could be detected in the first 2 days of the lag phase. mRNA production became maximal with 20 molecules per cell in the early exponential growth phase but then decreased to less than 10 molecules per cell. When TCB was depleted and the cells entered the stationary phase, the mRNA content decreased slowly below the detection limit within 4 days. In order to compare detection of tcbC mRNA in pure culture and in river sediment, cells of strain P51 pregrown on TCB were added to sediment and RNAs extracted. In sediment samples containing 5 x 10(8) cells per gram the tcbC mRNA was quantifiable by RT/PCR. The mRNA recovery was about 3% as compared to the inoculum. The detection limit of the RT/PCR method was about 10(7) mRNA molecules per gram sediment or 10(6) copies per ml culture medium which corresponded in our case to 10(5) molecules per reaction vial.