SENSITIVE DETECTION OF A NOVEL CLASS OF TOLUENE-DEGRADING DENITRIFIERS, AZOARCUS-TOLULYTICUS, WITH SMALL-SUBUNIT RIBOSOMAL-RNA PRIMERS AND PROBES

Azoarcus tolulyticus is a new class of widely distributed, toluene-deg rading denitrifiers of potential importance in remediating benzene, to luene, ethylbenzene, and gene (BTEX)-contaminated environments. To det ect these organisms in the environment, 16S rRNA gene-based phylogenet ic probes were developed. Two sets of specific PCR amplification prime rs and two oligonucleotide hybridization probes were designed and test ed against both closely and distantly related environmental isolates. All of these primers and probes were specific to the species A. toluly ticus. The sensitivity of the PCR amplification primer sets was evalua ted with DNA isolated from A. tolulyticus Tol-4 pure culture and from sterile soils seeded with a known number of Tol-4 and Escherichia call cells. These primer sets were able to detect 1 fg to 1 pg of template DNA from the pure culture and 1.11 x 10(2) to 1.1 x 10(8) Tol-4 cells per g of soil in the presence of 1.56 x 10(10) E. coli cells. These t wo PCR amplification primers were also successfully tested at two fiel d sites. The primers identified the A. tolulyticus strains among the t oluene-degrading bacteria isolated from a low-O-2-high-NO3- aquifer at Moffett Field, Calif. Also, the presence of A. tolulyticus was detect ed in the groundwater samples from a BTEX-contaminated aquifer at an i ndustrial site in Detroit, Mich., which showed anaerobic toluene degra dation.