Date of Award

2004

Degree Type

Dissertation

Department

Interdepartmental Program

First Advisor

Paul S. Cohen

Abstract

The Cra protein is a global regulator of carbon and energy metabolism for glycolysis and gluconeogenesis. The live oral vaccine candidate, Salmonella typhimurium SR-11 Fad, is unable to utilize gluconeogenic substrates as sole carbon sources and is avirulent and protective in BALB/c mice. Furthemore, the era gene is interrupted in this strain of Salmonella. The cra gene was suspected to be regulated by another global regulator, acetyl phosphate. To further investigate the regulation of the cra gene and to determine if gluconeogenesis is linked to virulence, mutations in the gluconeogenic genes fbp (fructose-1,6-bisphosphatase), maeB (NADP-dependent malic enzyme), and sfcA (NAD-dependent malic enzyme) were constructed in S. typhimurium SR-11. A mutation in the pta (phosphotransacetylase) gene was also constructed to interrupt acetyl phosphate synthesis. Virulence assays in BALB/c mice were performed with these mutant strains. A cra promoter-lacZ transcriptional fusion was also inserted into the chromosome of these mutant strains to assay for the era promoter activity during growth on various glycolytic and gluconeogenic substrates.

The SR-11 fbp, SR-11 maeB, SR-11 sfcA- mutant strains were virulent in BALB/c mice, whereas the pta mutant strain was avirulent. The mutation in the maeB gene slightly down-regulated the era promoter activity when grown on either gluconeogenic or glycolytic substrates versus the SR- 11 cradl craz integrant control strain. A mutation in either the sfcA gene or the pta gene significantly (P = 0.05) up-regulated the cra promoter activity for growth on both gluconeogenic and glycolytic substrates versus the control strain. This up-regulation of cra promoter activity, combined with their virulence in BALB/c mice, suggest that the sfcA-encoded NAD-dependent malic enzyme and acetyl phosphate act in concert as a repressor of the cra gene. It also appears that the cra gene may be subject to regulation by multiple regulatory proteins or multiple forms of regulation.

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