Recombinant plasmid conferring proline overproduction and osmotic tolerance

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Abstract
A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.