Molecular biology of anaerobic toluene and m-xylene oxidation in Azoarcus sp. strain T

Natural attenuation is a promising technology for remediation of gasoline-contaminated sites. Understanding the biochemistry and molecular biology of enzymes responsible for natural attenuation will improve our ability to predict and enhance natural attenuation. Biochemical studies revealed a novel fumarate addition reaction for initiation of anaerobic toluene oxidation. This reaction appears to be a common mode for initiation of anaerobic mineralization of a wide range of compounds. The molecular biology of anaerobic toluene and m-xylene oxidation in Azoarcus sp. strain T will be discussed in this work.; Azoarcus sp. strain T is a facultative denitrifying bacterium that anaerobically mineralizes both toluene and m-xylene. To facilitate genetic studies in this microorganism, a reliable transformation procedure was developed. Several broad host range vectors that are replicated in Azoarcus sp. strain T were identified, as well as a suicide vector system that allowed construction of chromosomal insertions and deletions.; The first step of the pathway for anaerobic oxidation of toluene, catalyzed by benzylsuccinate synthase, is the addition of toluene to fumarate to form benzylsuccinate. The genes encoding the three subunits of benzylsuccinate synthase were cloned and sequenced. Analysis of a null mutant in the gene encoding the largest subunit of benzylsuccinate synthase, bssA, showed that a single enzyme, benzylsuccinate synthase, is necessary for growth on both toluene and m-xylene. Reverse transcriptase-PCR studies, as well as Northern blot studies and transcriptional start site mapping, showed that the structural genes encoding benzylsuccinate synthase, bssCAB , as well as two other genes, named bssD and bssE, are transcribed as an operon. Transcriptional start sites were defined for the bssDCABE operon, as well as for an upstream operon containing two genes, tdiS and tdiR. To quantitatively determine bssDCABE expression under different conditions, a translational lacZ fusion to bssB was generated.; tdiS encodes a protein with homology to sensor kinases of two-component regulatory systems. A null mutant in the tdiS gene was constructed to determine its role in anaerobic toluene oxidation. The tdiS gene was heterologously expressed in Escherichia coli, and polyclonal antibodies to inclusion bodies of the TdiS protein were generated for analysis of expression of tdiS.