The lrp regulon of Escherichia coli: Mutational analysis of leucine-regulated genes
Abstract
The tdh promoter of Escherichia coli is induced seven- to eight-fold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression from the tdh promoter. These mutants were hypersensitive to scL-valine and to borrelidin, indicating a depleted internal threonine pool. The tdh-constitutive mutants also exhibited hypersensitivity to both scL-leucine and scD, scL-4-azaleucine, indicating a possible enhancement of leucine transport capability. Growth studies showed that one of the mutants exhibits a longer doubling time (101 minutes) than the isogenic wild type strain (81 minutes). The valine hypersensitive phenotype was used to map the mutation in these strains to a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25 bp region between coordinates $-$69 and $-$44 of the tdh promoter. Deletion analysis in an lrp strain and mutagenesis of a palindrome at coordinates $-$34 to $-$51, indicated that there are no major transcriptional regulators of the tdh promoter that act independently of Lrp. Unlike many Lrp-regulated genes, the expression of tdh was not affected by 100 mg/ml scL-alanine. Disruption of a 12 bp sequence upstream of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were identified in 10 of the 14 Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the Lrp-regulated serA promoter region was determined. No Lrp consensus sequence was present, suggesting that Lrp may act indirectly at this promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR, affects expression from both the tdh and serA promoters. The livR allele has been shown recently to be an allele of Lrp. Among all the E. coli K-12 sequences in the GenBank and EMBL databases, there were 420 Lrp consensus sequences. However 165 of these sequences lay in 123 promoter regions. Two promoters containing the Lrp consensus sequence, rbsDACBK and tyrP, were shown to be unaffected by leucine or Lrp.
Degree
Ph.D.
Advisors
Somerville, Purdue University.
Subject Area
Genetics|Molecular biology|Biochemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.