Regulation of Proline Utilization in Salmonella Typhimurium
Hahn, Donald Richard
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https://hdl.handle.net/2142/71194
Description
Title
Regulation of Proline Utilization in Salmonella Typhimurium
Author(s)
Hahn, Donald Richard
Issue Date
1988
Doctoral Committee Chair(s)
Maloy, Stanley R.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Genetics
Biology, Microbiology
Abstract
Salmonella typhimurium LT2 can utilize proline as a sole source of carbon or nitrogen. The two genes required for this process, putP and putA, are expressed divergently. The putA gene product is involved in a unique form of autoregulation. In the presence of the inducer, proline, the putA protein is active as a dehydrogenase. In the absence of proline, the putA protein represses transcription of the put operon. The purpose of this study was to characterize the regulatory sites involved in control of the put operon.
A large number of cis-dominant mutants were isolated which overexpress the put operon. These mutations define interactions of four classes of regulatory proteins at the put operon: RNA polymerase, CRP-cAMP, putA repressor, and nitrogen regulatory proteins.
Cis-dominant putP and putA promoter up mutations were isolated. Expression of put in these mutants retained catabolite repression and induction by proline, suggesting that the mutations affect the promoters which are regulated in vivo. Mapping these mutations indicated that the put promoters were convergent. The DNA sequence of the regulatory region, indicated that there is a single putA promoter, and three putP promoters. Transcription of putA requires CRP-cAMP activation, while the putP gene is transcribed at a low level even without CRP-cAMP. Two sequences in the put regulatory region are similar to the CRP consensus binding site.
Cis-dominant mutations which affect the operator constitutively expressed both genes. This indicates that a single operator site is required for repression of both genes. The operator mutations mapped between the putP and putA promoter mutations, in a region with a large palindrome which may function as the operator.
The wild type put operon was not believed to be under nitrogen control (ntr) in S. typhimurium, because proline cannot be used as a nitrogen source when the cells are catabolite repressed. However, putA expression in S. typhimurium was activated under nitrogen limiting conditions when an alternate nitrogen source was provided. Furthermore, some cis-dominant mutations which expressed putA independent of catabolite repression, also showed ntrC dependent expression of putA. The properties of these mutants indicate that nitrogen regulation of put takes place by a unique regulatory mechanism.
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