Nitrate metabolism in yeasts studies on regulation and properties of nitrate reductase in Candida utilis. Ph.D. Thesis

Abstract

Synopsis of the thesis entitled “NITRATE METABOLISM IN YEASTS: STUDIES ON REGULATION AND PROPERTIES OF NITRATE REDUCTASE IN CANDIDA UTILIS” submitted by V. P. Choudary Microbiology & Cell Biology Laboratory for the award of the Ph.D. degree of the Indian Institute of Science, Bangalore – 560012, India

Nitrate is one of the important sources of inorganic nitrogen for higher plants and many microorganisms. In contrast to the large number of plants, algae, fungi, and bacteria that can utilize nitrate, the number of nitrate positive yeasts is very small, especially among species of Candida. Although the ability to utilize nitrate has traditionally been employed as a major criterion in classifying yeasts, virtually no information is available on the biochemical basis of this wide variation (Chapter I). Hence, the following studies were undertaken on the assimilation of nitrate and related compounds by various species of Candida, Saccharomyces cerevisiae, and on the regulation of nitrate reductase in C. utilis. All six yeast species examined utilized ammonia but failed to grow readily on nitrate, except C. utilis. The intermediates (nitrite, hydroxylamine, and hydrazine) not only failed to support good growth of C. utilis but also interfered with ammonia utilization. Cell free preparations of C. utilis grown on nitrate showed both NAD(P)H:nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H:nitrite oxidoreductase (EC 1.6.6.4) activities. Nitrate reductase, the first enzyme of the nitrate assimilation pathway, was characterized in detail in vitro (Chapter II). A new method was developed to permeabilize induced yeast cells for assaying nitrate reductase in situ. The in situ assay was more sensitive than the in vitro method and did not require added cofactors. It greatly facilitated the study of various aspects of nitrate reductase regulation in C. utilis (Chapter III). Synthesis and maintenance of nitrate reductase in C. utilis showed an absolute requirement for both a suitable inducer and an energy source. Nitrates were the most effective inducers, followed by nitrites, D amino acids, and L phenylalanine. Experiments with cycloheximide and actinomycin D revealed that the enzyme was synthesized de novo, and that inducer presence was necessary for both enzyme synthesis and in vivo stability. Hydroxylamine and hydrazine exerted a strong repressive effect on nitrate reductase synthesis, followed in decreasing order by ammonia, urea, and glutamate. Nitrate reductase activity disappeared rapidly in vivo during nitrogen starvation and on exposure to ammonia (Chapter IV). Various properties of nitrate reductase from C. utilis were studied using partially purified preparations (Chapter V).