1887

Abstract

SUMMARY: Mutants of K12 with deletions in the region of the chromosome were obtained for use in studies on the expression of the (pyruvate dehydrogenase complex, specific components) and (lipoamide dehydrogeriase) genes. These were isolated by selecting spontaneous mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes. From 2892 isolates tested, the AroP- phenotypes of 2322 were confirmed and, of these, 28 stable and independently-derived auxotrophs were designated as deletion mutants.

Six nutritionally-distinct categories were recognized: Nad- (8 strains); NadAce (7); Nad ‘Ace’ (3); Ace (8); ‘Ace’ (1); Lpd (1). The Ace phenotypes of four isolates designated ‘Ace’ were leaky and enzymological studies confirmed that they had less than 7 % of parental pyruvate dehydrogenase complex activity.

Enzymological studies showed that the 15 Ace or NadAce strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (Eip) activities and only three retained detectable dihydrolipoamide acetyltransferase (E2p). The one Lpd- strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and α-ketoglutarate dehydrogenase complexes.

The results confirmed the gene order and indicated that no other essential functions are determined by genes within the region. Resistance to lactate during growth of mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex. None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex. Six mutants having Ace or ‘Ace’ phenotypes were more sensitive than the parental strains and expression of their operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities.

The lipoamide dehydrogenase activities of the deletion strains (Lpd) ranged between 30 % and 100 % of parental levels indicating that expression of the gene may be affected by the operon but can be independent.

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/content/journal/micro/10.1099/00221287-99-2-263
1977-04-01
2019-10-14
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