Li, Jiping2006-07-282006-07-2819991999http://hdl.handle.net/10012/454Enterobacter cloacae UW4 and CAL2 produce an inducible l-aminocyclopropane-1-carboxylate (ACC) deaminase that hydrolyzes ACC into et-ketobutyrate and ammonia. The genes encoding the ACC deaminases, i.e. acdS, were cloned from both species of E. cloacae and sequenced. The deduced amino acid sequences of the ACC deaminases are highly homologous to previously reported pseudomonad ACC deaminase sequences (95-99% identity) and the cloned genes can be expressed in E.coli cells. When the gene was introduced into other soil bacteria, these strains gained the ability to grow on ACC as a sole source of nitrogen and to promote canola root elongation. It was found that the regulation of the acdS gene in strain UW4 requires both the product of an ORF (upstream of acdS) and ACC. The ORF was identified as an Lrp-like protein containing a helix-turn-helix motif. The results from tra11s complementation experiments showed that the protein encoded by the ORF behaves in a manner similar to known Lrp proteins to positively regulate the acdS promoter. Using illxAB reporter genes as promoter-probes, in addition to ACC, L-alanine also induced acdS gene expression, albeit at a low level. To evaluate the role of ACC deaminase in plant growth promoting rhizobacteria (PGPR), a model has been proposed in which PGPR stimulate plant growth as a consequence of their production of ACC deaminase which causes a lowering of plant ethylene levels resulting in longer roots. In an attempt to assess the model, an ACC deaminase mutant was created by targeted gene replacement and has been shown to be completely deficient in its ability to use ACC as a nitrogen source, devoid of ACC deaminase activity and in contrast to the wild-type E. cloacae UW4, unable to promote canola root elongation in gnotobiotic growth pouches. This result suggests that ACC deaminase plays a direct role in the promotion of plant growth as described in the model. In addition, a novel gene from strain UW4 with the ability to hydrolyze ACC has been isolated and partially characterized. Comparison studies of the novel gene with other similar genes suggests that this gene may have evolved from the primitive protein common to the amidohydrolase superfamily of proteins, and that this gene encodes a distinct type of ACC deaminase.application/pdf11415111 bytesapplication/pdfenCopyright: 1999, Li, Jiping. All rights reserved.Harvested from Collections CanadaDoctoral Thesis