Mutants of Escherichia coli K12 Permeable to Haemin

A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.

Section: Methodsmentioning

confidence: 99%

Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity

Page

Ferguson

1994

“…Hematin was prepared from hemin (Sigma) as described previously (28) and was used at a final concentration of 20 jig/ml.…”

Section: Methodsmentioning

confidence: 99%

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium

Elliott

1993

In a previous search for mutants of Salnonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of 1-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

“…The lack of detectable heme coupled with failure of the heme precursor delta-aminolevulonic acid to restore the wild-type phenotype to strain HS18A suggested that the genetic lesion in the mutant is involved in the latter stages of heme biosynthesis. Such mutants have been observed (26,31) and reportedly do not respond to exogenous heme supplied in the medium (31), an observation consistent with the growth characteristics of strain HS18A. Genetic mapping.…”

Section: Resultsmentioning

confidence: 49%

“…Reconstitution experiments have demonstrated that the inactive apoforms of these respiratory components are synthesized even in the absence of heme biosynthesis (12,26). E. coli mutants impaired in heme biosynthesis have been difficult to isolate since this organism is reportedly impermeable to hemin (31).…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of respiratory-deficient mutants of Escherichia coli K-12

Schellhorn

Hassan

1988

Several mutants ofEscherichia coli K-12 Moody, J. Biol. Chem., in press). Though lesions in both aerobic and anaerobic respiratory pathways have been described (for a review, see reference 13), the effect of such mutations on the regulation of enzymes involved in aerobic survival has received little attention.In this work, we isolated mutants which were unable to grow aerobically on media devoid of a source of fermentable carbohydrate. Changes in the aerobic metabolism and heme and cytochrome content were evaluated in one of the mutants. We also examined aerobic induction of MnSOD, catalase, and peroxidase in this mutant. The results of these studies form the body of this report.