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Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

ABSTRACT: In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective PnuA- phenotype. These mutants retain regulation properties but are unable to transport nicotinamide mononucleotide (NMN) intact across the cell membrane. Other nadR mutants lose both regulatory and transport capabilities, while a third class loses only regulatory ability. The unusual NMN transport activity requires both the PnuC and NadR proteins, with the pnuC locus residing in an operon with nadA. To prove that nadR encoded a single protein and to gain insight into a regulatory target locus, the nadR and nadA pnuC loci were cloned and sequenced. A DNA fragment which complemented both regulatory and transport mutations was found to contain a single open reading frame capable of encoding a 409-amino-acid protein (47,022 daltons), indicating that NadR is indeed bifunctional. Confirmation of the operon arrangement for nadA and pnuC was obtained through the sequence analysis of a 2.4-kilobase DNA fragment which complemented both NadA and PnuC mutant phenotypes. The nadA product, confirmed in maxicells, was a 365-amino-acid protein (40,759 daltons), while pnuC encoded a 322-amino-acid protein (36,930 daltons). The extremely hydrophobic (71%) nature of the PnuC protein indicated that it was an integral membrane protein, consistent with its central role in the transport of NMN across the cytoplasmic membrane. The results presented here and in previous studies suggest a hypothetical model in which NadR interacts with PnuC at low internal NAD levels, permitting transport of NMN intact into the cell. As NAD levels increase within the cell, the affinity of NadR for the operator regions of nadA, nadB, and pncB increases, repressing the transcription of these target genes.

SUBMITTER: Foster JW

PROVIDER: S-EPMC213241 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

Foster J W JW Park Y K YK Penfound T T Fenger T T Spector M P MP

Journal of bacteriology 19900801 8

In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective P ...[more]

PMID: 2198247

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Project description:In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.

Dataset Information

Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

Publications

Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

Similar Datasets

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