AN ANALYSIS OF THE HISU REGULATORY MUTATION IN SALMONELLA TYPHIMURIUM: ALTERED STABLE RNA SYNTHESIS AND THE PLEIOTROPIC EFFECTS ON ILV REGULATION

DONELLA JOYCE WILSON, Purdue University

Abstract

The regulatory mutation hisU1820 in Salmonella typhimurium was originally isolated by Roth, Anton, and Hartman in an effort to identify mutants derepressed for the histidine operon. Subsequently, the results of studies showing altered regulation of both isoleucine and valine biosynthesis and altered RNA synthesis served to clearly establish the pleiotropic nature of this mutant. To gain an increased understanding of this mutation, the study reported herein focuses on the control of acetohydroxy acid synthase (AHAS) formation and the effects of a nutritional shiftdown on stable RNA accumulation with respect to cellular levels of guanosine polyphosphates. In addition to a four-fold increase of his enzyme levels, this hisU mutant exhibits a four-fold reduction in the expression of the ilvGEDA operon with a concomitant several-fold increase in the expression of AHAS valine-sensitive activity (AHAS('s)) when grown in minimal medium. Further investigation of ilv regulation revealed that upon the addition of isoleucine to cells previously grown in valine, there is a burst of AHAS('s) activity. It was concluded that isoleucine was a positive effector on AHAS('s) expression and that the hisU mutation accentuates the isoleucine-effect, which was found to be transient over three to four mass doublings and to be dependent on protein synthesis. The aforementioned altered regulation in the expression of amino acid biosynthetic operons in hisU stimulated studies on this mutation's effect on the control of stable RNA synthesis (rRNA and tRNA). We observed that during a NB (--->) Glc downshift, the rate of RNA accumulation in hisU was approximately four-fold greater than that in the wild type, with a predominant overproduction of small RNA species. During similar types of metabolic shifts, substantial increases in the levels of ppGpp and of pppGpp were observed in hisU. It is of interest that both hisU and its wild type are relA('+), while only the hisU mutant expresses a relaxed phenotype during nutritional transitions. This altered phenotype is observed, in spite of significant amounts of ppGpp accumulated in the cell. A model for the regulatory alterations occasioned by the hisU mutation rests on the central premise that a gene which codes for some element that promotes increased stabilization of stable RNAs is altered in this mutant. The correspondent increases in guanosine polyphosphates are, therefore, compensatory to the noted increases in RNA accumulation in the hisU mutant post metabolic transitions. Our conclusions from the results presented further implicate the involvement of the hisU gene product in a general regulatory control mechanism essential to macromolecular adjustments of coupled catabolic, biosynthetic, and polymerization pathways, occasioned by changing cellular environments.

Degree

Ph.D.

Subject Area

Molecular biology

COinS