Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8α(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.
BergD. E.1977; Insertion and excision of the transposable resistance determinant Tn5. In DNA Insertion Elements, Plasmids and Episomes pp
BukhanA. I.,
ShappiroJ. A.,
AdhyaS. L.
Edited by Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
BergD. E.,
JorgensonR.,
DaviesJ.1978; Transposable kanamycin-neomycin resistance determinants. In Microbiology-1978, pp 13–15SchlesingerD.
Edited by Washington, DC: American Society for Microbiology;
BossiL.,
CiampiM. S.1981; DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella. Molecular and General Genetics 183:406–408
BradfordM. M.1976; Sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
CammackR.,
PatilD. S.,
WeinerJ. H.1986; Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S] cluster. Biochimica et biophysica acta 870:538–544
CecchiniG.,
AckrellB. A. C,
DeshlerJ. O.,
GunsalusR. P.1986; Reconstitution of quinone reduction and characterization of Escherichia colifumarate reductase activity. Journal of Biological Chemistry 261:1808–1814
ClewellD. B.,
HelinskiD. R.1972; Effect of growth conditions on the formation of the relaxation complex of supercoiled ColEl deoxyribonucleic acid and protein in Escherichia coli. Journal of Bacteriology 110:1135–1146
ColeS. T.1982; Nucleotide sequence coding for the flavoprotein subunit of the fumarate reductase of Escherichia coli. European Journal of Biochemistry 122:479–484
ColeS. T.,
GrundströmT.,
JaurinB.,
RobinsonJ. J.,
WeinerJ. H.1982; Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli. European Journal of Biochemistry 126:211–216
ColeS. T.,
CondonC.,
LemireB. D.,
WeinerJ. H.1986; Molecular biology, biochemistry and bioenergetics of fumarate reductase, a complex membrane-bound iron-sulfur flavoenzyme. Biochimica et biophysica acta 811:381–403
DeBruijnF. J.,
AusubelF. M.1981; The cloning and transposon Tn5 mutagenesis of the ginA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nil and hutoperons. Molecular and General Genetics 183:289–297
DeBruijnF. J.,
StrokeI. L.,
MarvelD. J.,
AusubelF. M.1983; Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis. EMBO Journal 2:1831–1838
DickieP.,
WeinerJ. H.1979; Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli. Canadian Journal of Biochemistry 57:813–821
GoldbergA. L.1972; Degradation of abnormal proteins in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 69:422–426
GrundströmT.,
JaurinB.1982; Overlap between ampC and frd operons on the Escherichia colichromosome. Proceedings of the National Academy of Sciences of the United States of America 79:1111–1115
HederstedtL.1983; Succinate dehydrogenase mutants of Bacillus subtilis lacking covalently bound flavin in the flavoprotein subunit. European Journal of Biochemistry 132:589–593
HederstedtL.,
MaguireJ. J.,
WaringA. J.,
OhnishiT.1985; Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase. Journal of Biological Chemistry 260:5554–5562
JohnsonR. C.,
YinJ. C. P,
ReznikoffW. S.1982; Control of Tn5 transposition in Escherichia coii is mediated by protein from the right repeat. Cell 30:873–882
JohnsonD. A.,
GautschJ. W.,
SportsmanJ. R.,
ElderJ. H.1984; Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Analysis Techniques 1:3–8
LemireB. D.,
RobinsonJ. J.,
WeinerJ. H.1982; Identification of the membrane anchor polypeptides of Escherichia coli. Journal of Bacteriology 152:1126–1131
LemireB. D.,
RobinsonJ. J.,
BradleyR. D.,
ScrabaD. G.,
WeinerJ. H.1983; Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli. Journal of Bacteriology 155:391–397
LohmeierE.,
HagenD. S.,
DickieP.,
WeinerJ. H.1981; Cloning and expression of the fumarate reductase gene of Escherichia coli. Canadian Journal of Biochemistry 59:158–164
LupskiJ. R.,
SmillieB. L.,
BlattnerF. R.,
GodsonG. N.1982; Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis. Molecular and General Genetics 185:120–128
MillerJ. H.,
CalosM. P.,
GalasD.,
HoferM.,
BuchelD. E.,
Muller-HillB.1980; Genetic analysis of transpositions in the lac region of Escherichia coli. Journal of Molecular Biology 144:118
PannekoekH.,
HillieJ.,
NoordermeerI.1980; Relief of polarity caused by transposon Tn5: application in mapping a cloned region of the Escherichia coli uvrB locus essential for UV resistance. Gene 12:51–61
RenartJ.,
ReiserJ.,
StarkG. R.1979; Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proceedings of the National Academy of Sciences of the United States of America 76:3116–3120
RobinsonJ. J.,
WeinerJ. H.1981; The effects of anions on fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli. Biochemical Journal 199:473–477
RobinsonJ. J.,
WeinerJ. H.1982; Molecular properties of fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli. Canadian Journal of Biochemistry 60:811–816
SinghA. P.,
BraggP. D.1975; Reduced nicotinamide adenine dinucleotide dependent reduction of fumarate coupled to membrane energization in a cytochrome deficient mutant of Escherichia coliK12. Biochimica et biophysica acta 396:229–241
SpencerM. E.,
GuestJ. R.1974; Proteins of the inner membrane of Escherichia coli: changes in composition associated with anaerobic growth and fumarate reductase amber mutation. Journal of Bacteriology 117:954–959
UndenG.,
KrögerA.1981; The function of the subunits of the fumarate reductase complex of Vibrio succinogenes. European Journal of Biochemistry 120:577–584
WeinerJ. H.,
DickieP.1979; Fumarate reductase of Escherichia coli Elucidation of the covalent flavin component. Journal of Biological Chemistry 254:8590–8593
WeinerJ. H.,
LemireB. D.,
ElmesM. L.,
BradleyR. D.,
ScrabaD. G.1984; Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle. Journal of Bacteriology 158:590–596
WeinerJ. H.,
CammackR.,
ColeS. T.,
CondonC.,
HonoréN.,
LemireB. D.,
ShawG.1986; A novel mutant of Escherichia coli fumarate reductase decoupled from electron transport. Proceedings of the National Academy of Sciences of the United States of America 83:2056–2060
YamatoI.,
AnrakuY.,
HirosawaK.1975; Cytoplasmic membrane vesicles of Escherichia coliI. A simple method for preparing the cytoplasmic and outer membranes. Journal of Biochemistry 77:705–718