Burt D. Ensley

Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4.

The multicomponent enzyme, naphthalene dioxygenase, initiates the metabolism of naphthalene by Pseudomonas putida strains G7 (PpG7) and NCIB 9816-4 (Pp9816-4). The genes involved (nahAaAbAcAd) are encoded by the NAH7 and pDTG1 plasmids, respectively, and form part of the nah operon. The locations of the structural genes were determined on previously cloned fragments of DNA. The nucleotide (nt) sequences were determined for nahAaAb from Pp9816-4 and for nahAaAbAcAd from PpG7. The appropriate open reading frames were identified using N-terminal amino acid sequences determined from the purified proteins. The two nt sequences showed 93% homology, with the least homology seen upstream from the promoter region.

A T7 RNA polymerase-based system for the construction of Pseudomonas strains with phenotypes dependent on TOL-meta pathway effectors.

A general method to construct recombinant Pseudomonas putida (and related bacteria), which transcribe specific genes inserted into their chromosome in response to the presence of alkyl- and halobenzoates, has been developed. The system is based on the ability of the T7 RNA polymerase (T7pol) to initiate transcription from cognate promoter sequences located upstream from cloned genes. A specialized transposon, mini-Tn5 xylS/Pm::T7pol, was constructed which contains the structural T7 gene 1 downstream from the XylS protein/benzoate-regulated Pm promoter of the meta-operon of the TOL catabolic plasmid. This transposon was stably inserted into the chromosome of a P. putida target strain so that gene 1 is transcribed upon exposure of the bacteria to benzoate effectors of the XylS regulator. Genes whose expression is to be mediated by T7pol are cloned in mini-Tn5 transposons containing T7 promoter sequences upstream from the cloning site and then the hybrid transposons are inserted into different positions in the same chromosome. In this way, expression of the genes cloned within the mini-Tn5 vectors is dependent on the T7pol/XylS/Pm system. This expression device is particularly well suited for applications in which the expression of two or more genes is to take place in response to a single chemical signal, i.e., exposure to certain aromatic compounds.