The metabolic pathway for the reduction and incorporation of sulphate in Paracoccus denitrificans strain NCIB 8944 has been elucidated and the control and regulation of the pathway is reported. Several enzymes of the sulphate metabolic pathway have been assayed in P. denitrificans grown on different substrates. In addition, several enzymes have been purified and in vitro inhibitor studies conducted on them. Cysteine plays a primary role in the control of sulphur metabolism in P. denitrificans.
We have previously described a large family of mutants of Klebsiella aerogenes which were selected by continuous culture on xylitol and which superproduce ribitol dehydrogenase. One of these strains was found to harbour a high copy number 2.1 × 106 dalton plasmid. This plasmid is a deletion derivative of a low copy number 3.5 × 106 dalton plasmid present in the ancestral strain of K. aerogenes. However, since these plasmids do not contain the genes required for pentitol catabolism and some enzyme-superproducing strains have lost all DNA homologous to the plasmids, they are not implicated in the fast growth on xylitol. The plasmids contain regions of homology with the Escherichia coli plasmid ColE1.
The phenotypes of certain mutant strains of Pseudomonas aeruginosa were reported to be pleiotropic for nitrate reduction; these strains were selected for their inability to dissimilate nitrate and were found also to have lost the ability to assimilate nitrate. We now report that the isolation procedure selected two mutations, one in genes encoding the synthesis of dissimilatory nitrate reductase (narA, narB or narE) and another in one of the genes (nas) encoding the synthesis of assimilatory nitrate reductase. Thus in P. aeruginosa dissimilatory and assimilatory nitrate reductases are genetically distinct. However, a loss of both enzymes is necessary to prevent slow dissimilatory growth on nitrate. Assimilatory nitrate reductase requires molybdenum to function, as does dissimilatory nitrate reductase. Lesions in narD affect incorporation of molybdenum into both enzymes, and hence exert a pleiotropic effect.
The recombinant plasmids RSF2124-trp and pSC101-trp were examined for their phenotypic stability in Escherichia coli W3110 and its derivatives under various culture conditions. RSF2124-trp and pSC101-trp were stable in a trpAE1 strain. In an amber mutant of the tryptophan repressor gene, RSF2124-trp was fairly stable, whereas pSC101-trp was unstable. All Trp- segregants from the pSC101-trp carrier had lost the entire plasmid. In a mutant carrying the tnaA mutation, RSF2124-trp was unstable in rich media. Most Trp- segregants that appeared under these conditions were deleted in trp genes as well as in the cI gene on the recombinant plasmid. pSC101-trp in this tnaA mutant was also unstable. All Trp- segregants had lost the plasmid. Studies of enzyme activities revealed that the greater the activity of anthranilate synthase and tryptophan synthase in bacteria, the more segregants tended to appear in the stability test. RSF2124 and pSC101 without the trp gene were completely stable in the same bacteria. The apparent instability of bacteria carrying the recombinant plasmid could be explained by the lower growth rate compared with bacteria carrying only the vector plasmid, resulting in the enrichment of Trp- bacteria during culture.