The locus coding for the 3-nitrobenzoate dioxygenase of Comamonas sp. strain JS46 is flanked by IS1071 elements and is subject to deletion and inversion events.

In Comamonas sp. strain JS46, 3-nitrobenzoate (3Nba) is initially oxidized at the 3,4 position by a dioxygenase, which results in release of nitrite and production of protocatechuate. The locus coding for the 3Nba dioxygenase (designated mnb, for m-nitrobenzoate) was mobilized from strain JS46 using a plasmid capture method, cloned, and sequenced. The 3Nba dioxygenase (MnbA) is a member of the phthalate family of aromatic oxygenases. An open reading frame designated mnbB that codes for an NAD(P)H-dependent class IA aromatic oxidoreductase is downstream of mnbA. MnbB is tentatively identified as the oxidoreductase that transfers reducing equivalents to MnbA in strain JS46. The mnb locus is flanked by IS1071 elements. The upstream element is interrupted by a novel insertion sequence designated ISCsp1, and the transposase genes of the flanking insertion elements are transcribed in the direction opposite the direction of mnbA transcription. Spontaneous deletion of mnb occurs because of homologous recombination between the directly repeated flanking IS1071 elements. In addition, in approximately 0.007 to 0.2% of any population of JS46 cells growing on 3Nba, alternative orientations of mnb relative to the flanking IS1071 elements are detected. These alternative forms are the result of inversions of mnb and the flanking IS1071 elements. Inversions appear to occur because of homologous recombination between the inverted repeats that flank the IS1071 elements.

Predicting survival of a genetically engineered microorganism, Pseudomonas chlororaphis 3732RN-L11, in soil and wheat rhizosphere across Canada with linear multiple regression models.

Pseudomonas chlororaphis 3732RN-L11 survival rates in soil and wheat rhizosphere were measured using intact soil core microcosms representing 23 sites across Canada. Linear multiple regression (LMR) models were developed to predict the survival rate of this genetically engineered microorganism (GEM) as a function of soil parameters measured at the time of microcosm inoculation. LMR models were tested by comparing their predicted survival rates with observed survival rates from environmental introductions of the GEM by Gagliardi et al. (2001) at five field sites across Canada over two years. No soil parameter (e.g., % clay) was highly correlated with GEM survival rates in soil or wheat rhizosphere. Total fungal colony-forming units (CFUs), % soil titanium (positive correlations), and % soil magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rates in soil over two years. Total soil bacterial CFUs, nitrate, % soil potassium (positive correlations), and exchangeable magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rate in wheat rhizosphere over two years. While LMR models were statistically significant, they were unable to reliably predict the survival rate of the GEM in field trial introductions. The results indicate that there can be considerable uncertainty associated with predicting GEM survival for multi-site environmental introductions.

Comamonas testosteroni BR6020 possesses a single genetic locus for extradiol cleavage of protocatechuate.

A key intermediate for biodegradation of various distinct aromatic growth substrates in Comamonas testosteroni is protocatechuate (Pca), which is metabolized by the 4,5-extradiol (meta) ring fission pathway. A locus harbouring genes from C. testosteroni BR6020 was cloned, dubbed pmd, which encodes the enzymes that degrade Pca. The identity of pmdAB, encoding respectively the alpha- and beta-subunit of the Pca ring-cleavage enzyme, was confirmed by N-terminal sequencing and molecular mass determination of both subunits from the separated enzyme. Disruption of pmdA resulted in a strain unable to grow on Pca and a variety of aromatic substrates funnelled through this compound (m- and p-hydroxybenzoate, p-sulfobenzoate, phthalate, isophthalate, terephthalate, vanillate, isovanillate and veratrate). Growth on benzoate and o-aminobenzoate (anthranilate) was not affected in this strain, indicating that these substrates are metabolized via a different lower pathway. Tentative functions for the products of other pmd genes were assigned based on sequence identity and/or similarity to proteins from other proteobacteria involved in uptake or metabolism of aromatic compounds. This study provides evidence for a single lower pathway in C. testosteroni for metabolism of Pca, which is generated by different upper pathways acting on a variety of aromatic substrates.