Genetic dissection of the functional domains of the PutA protein of Salmonella typhimurium
Proline catabolism is a common function in both prokaryotes and in the mitochondria of eukaryotic cells. In Salmonella typhimurium and Escherichia coli proline can be utilized as a sole source of carbon, nitrogen or energy. Proline utilization (put) requires the expression of the two genes of the put operon: the putP gene, which encodes proline permease, and the putA gene, which encodes a multifunctional membrane-associated dehydrogenase that degrades proline to glutamate. PutA protein also autogenously regulates transcription of the put operon. The deduced amino acid sequence of PutA exhibits similarities to eukaryotic protein kinases and to other dehydrogenases which catalyze reactions similar to those of PutA. Induction of the put operon requires proline, oxygen, or another terminal electron acceptor, and available membrane binding sites. The following model explains the regulation: under inducing conditions, PutA binds to the membrane where it is enzymatically active, but when the intracellular concentration of proline is low or when functional membrane binding sites are limiting, PutA accumulates in the cytoplasm where it represses transcription of the put operon. This unique autoregulatory mechanism is mediated by the cellular location of PutA and allows induction of the put operon only when both the inducer, high intracellular proline concentration, and functional membrane sites required for enzyme activity are available. Both enzymatic functions of PutA (proline dehydrogenase and P5C dehydrogenase activity) are apparently required for induction of the put operon. However, only proline dehydrogenase activity appears to be necessary for membrane association of PutA. Full induction of putA expression requires that the synthesized PutA be enzymatically active, thus avoiding the wasteful synthesis of PutA if either membrane sites or proline (which are both required for enzymatic activity) are not available. Mutations decreasing the rate of either enzymatic activity of PutA result in decreased put expression, suggesting that PutA senses the enzymatic activity of the protein. Autogenous regulation of the putA gene may be physiologically important since PutA overexpression, like that of many membrane-associated proteins, is lethal. Thus, by shutting off its own synthesis when membrane sites are saturated or the protein is not enzymatically active, PutA may avoid lethal overexpression.
Biology, Molecular, Biology, Microbiology
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign