Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12

Abstract

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21,202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription-translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker forAgrobacterium tumefaciens-Mediated Transformation of Maize

“…Wild-type E. coli strains showed no sensitivity to butafenacil at any concentration, consistent with the reported resistance of the native bacterial enzyme to similar herbicides (Sasarman et al, 1993). In contrast, the E. coli strain SASX38 complemented by either Arabidopsis PPO-1 or PPO-2 was clearly sensitive to butafenacil, with strong growth inhibition at concentrations as low as 10 nm.…”

“…Arabidopsis PPO cDNAs were isolated by functional complementation of the E. coli PPO mutant SASX38 (Sasarman et al, 1993). This bacterial strain Figure 1.…”

“…Complementation of E. coli hemG mutants has proved to be a routinely successful method for the isolation of eukaryotic PPO genes (Dailey et al, 1995;Nishimura et al, 1995;Narita et al, 1996; Lermontova et al, 1997) despite the fact that there is no sequence similarity between the hemG protein (Sasarman et al, 1993) and either plant or mammalian PPO enzymes. The ability to complement bacteria with plant enzymes also provides a method of isolating herbicide-tolerant variants of these enzymes.…”

“…PPO genes have been isolated from Escherichia coli (Sasarman et al, 1979(Sasarman et al, , 1993, Bacillus subtilis (Dailey et al, 1994), and plants (Narita et al, 1996;Lermontova et al, 1997;Ward and Volrath, 1998;Volrath et al, , 2000. Attempts have been made to improve plant tolerance to PPO inhibitory herbicides both by overexpressing native PPO genes in plants and by selecting for resistant mutants.…”

Dual Targeting of Spinach Protoporphyrinogen Oxidase II to Mitochondria and Chloroplasts by Alternative Use of Two In-frame Initiation Codons

“…In all instances, Protox requires a flavin cofactor for enzymatic activity and a detergent to be extracted from the membrane. Protox genes or cDNAs have been cloned from Escherichia coli (7), Bacillus subtilis (8), human (9), cow (5), mouse (10), and yeast (11). The calculated molecular masses of these Protox gene products range from 50 to 60 kDa, except for the 21-kDa E. coli.…”