The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.
ArmstrongS.K.,
PettisG.S.,
ForresterL.J.,
McIntoshM.A.1989; The Escherichia coli enterobactin biosynthesis gene, entD:nucleotide sequence and membrane localization of its protein product.. Mol Microbiol 3:757–766
BayerM.E.,
BayerM.H.,
LunnC.A.,
PigietV.P.1987; Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli.. J Bacteriol 169:2659–2666
BorchertS.,
StackelhausT.,
MarahielM.A.1994; Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis.. J Bacteriol 176:2458–2462
BowdenG.A.,
BaneyxF.,
GeorgiouG.1992; Abnormal fractionation of β-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.. J Bacteriol 174:3407–3410
BradfordM.M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.. Anal Biochem 72:248–254
BryceG.F.,
BrotN.1972; Studies on the enzymatic synthesis of the cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine in Escherichia coli.. Biochemistry 9:1708–1715
CoderreP.E.,
EarhartC.F.1984; Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes.. FEMS Microbiol Lett 25:111–116
DermanA.I.,
PuzissJ.W.,
BassfordP.J.JrBeckwithJ.1993; A signal sequence is not required for protein export in prlA mutants of Escherichia coli.. EMBO J 12:879–888
EarhartC.F.1987; Ferrienterobactin transport in Escherichia coli.
. In Iron Transport in Microbes, Plants and Animals pp. 67–84WinkelmannG.,
van der HelmD.,
NeilandsJ.B.
Edited by Weinheim:: VCH Verlagsgesellschaft.;
El YaagoubiA.,
KohiyamaM.,
RicharmeG.1994; Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.. J Bacteriol 176:7074–7078
GreenwoodK.T.,
LukeR.K.J.1976; Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin.. Biochim Biophys Acta 454:285–297
GreenwoodK.T.,
LukeR.K.J.1980; Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12, Salmonella typhimurium and Klebsiella pneumoniae. Physical association of enterochelin synthetase components in vitro.. Biochim Biophys Acta 614:185–195
GrossmanT.H.,
TuckmanM.,
EllestadS.,
OsburneM.S.1993; Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfp0 and Escherichia coli entD genes.. J Bacteriol 175:6203–6211
GuilvoutI.,
Mercereau-PuijalonO.,
BonnefoyS.,
PugsleyA.P.,
CarnielE.1993; High molecular weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis.. J Bacteriol 175:5488–5504
LangmanL.,
YoungI.G.,
FrostG.E.,
RosenbergH.,
GibsonF.1972; Enterochelin system of iron transport in Escherichia coli:mutations affecting ferric-enterochelin esterase.. J Bacteriol 112:1142–1149
LaVallieE.R.,
DiBlasioE.A.,
KovacicS.,
GrantK.L.,
SchendelP.F.,
McCoyJ.M.1993; A thioredoxin gene fusion expression system that circumvents inclusion body formation in the Escherichia coli cytoplasm.. Bio/Technology 11:187–192
LugtenbergB.,
MeijersJ.,
PetersR.,
van der HoekP.,
van AlphenL.1975; Electrophoretic resolution of the major outer membrane protein of Escherichia coli into four bands.. FEBS Lett 58:254–258
McIntoshM.A.,
EarhartC.F.1977; Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia coli.. J Bacteriol 131:331–339
NeilandsJ.B.1981b; Microbial iron transport compounds (siderophores) as chelating agents.. In Development of Iron Chelators for Clinical Use pp. 13–31MartellA.E.,
AndersonW.F.,
BadmanD.G.
Edited by Amsterdam:: Elsevier.;
RusnakF.,
SakaitaniM.,
DrueckhammerD.,
ReichertJ.,
WalshC.T.1991; Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.. Biochemistry 30:2916–2927
SchlumbohmW.,
SteinT.,
UllrichC.,
VaterJ.,
KrauseM.,
MarahielM.A.,
KruftV.,
Wittman-LieboldB.1991; An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.. J Biol Chem 266:23135–23141
TurgayK.,
KrauseM.,
MarahielM.A.1992; Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes.. Mol Microbiol 6:529–546
ViswanathaT.,
SzezepanE.W.,
MurrayG.J.1987; Bio-synthesis of aerobactin: enzymological and mechanistic studies.. In Iron Transport in Microbes, Plants and Animals pp. 117–132WinkelmannG.,
van der HelmD.,
NeilandsJ.B.
Edited by Weinheim:: VCH Verlagsgesellschaft.;
WalshC.T.,
LiuJ.,
RusnakF.,
SakaitaniM.1990; Molecular studies on enzymes in chorismate metabolism and the enterobactin biosynthetic pathway.. Chem Rev 90:1105–1129
WildJ.,
RossmeisslP.,
WalterW.A.,
GrossC.A.1996; Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli.. J Bacteriol 178:3608–3613
WoodrowG.C.,
YoungI.G.,
GibsonF.1975; Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.. J Bacteriol 124:1–6
WoodrowG.C.,
YoungI.G.,
GibsonF.1979; Biosynthesis of enterochelin in Escherichia coli K-12. Separation of the polypeptides coded for by the entD, E, F and G genes.. Biochim Biophys Acta 582:145–153