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 5U10V 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 U mRNA molecules per gram sediment or 10 T copies per ml culture medium which corresponded in our case to 10 S molecules per reaction vial. z

Influence of Temperature and Predation on Survival of Salmonella enterica Serovar Typhimurium and Expression of invA in Soil and Manure-Amended Soil

“…In the present study, we were only able to detect invA mRNA in samples with more than 10 6 cells per g of soil (detected by plate counting), and this detection limit is higher than what has been observed in previous studies on mRNA quantification in soil samples (26,37,40). The fact that the number of invA transcripts in general was lower than the invA gene copy numbers may indicate that invA expression is downregulated as a response to stress due to suboptimal conditions in the environmental sample.…”

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

“…The chlorocatechols formed by these two enzymes are then metabolized by an inducible chlorocatechol pathway to Krebs cycle intermediates (32). The inducer of the gene coding for chlorocatechol 1,2-dioxygenase is very likely to be a chloro-cis,cis-muconate formed from the corresponding chlorocatechol (24,25). One explanation of the observed multiphasic kinetics of transformation of 1,2,4-TCB by strain PS14 could be this different expression of the chlorobenzene-degrading enzymes.…”

Detection and Quantification of Gene Expression in Environmental Bacteriology

“…It has been applied to quantification of chloroaromatic-degrading Pseudomonas species strain B13 in marine water or sediment (53), genetically tagged cyanobacteria in Baltic Sea sediment (62), and ammonia-oxidizing Proteobacteria in compost (49). cRT-PCR techniques have also been used to quantify tcbC gene expression involved in 1,2,4-trichlorobenzene degradation (59), toxigenesis in the aquatic pathogen C. botulinum type E (58,82), and amoA expression levels in Nitrosomonas oligotropha and Nitrospira species (28). An advanced cRT-PCR assay has also been used with real-time technology to determine copy levels of the carbazole 1,9a-dioxygenase gene in Pseudomonas species (100).…”

“…The resurgence of the application of molecular tools for expression analysis from environmental sources is generally seen as a consequence of reduced technology costs, together with more effective methods for recovery of nucleic acids from environmental samples (83) and increasing amounts of data on the quantitative systems that are available. Applications are wide ranging, from quantitative analysis of rRNA genes in microbial communities (10) to detection of specific pathogens (54,55) and quantification of specific genes involved in biodegradative processes (59).…”