In the biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin, the small ORF cloY encodes a 71 aa protein which shows significant sequence similarity to mbtH from the mycobactin biosynthetic gene cluster of Mycobacterium tuberculosis. mbtH-like genes are frequently found in the biosynthetic gene clusters of peptide antibiotics and siderophores, but their function has remained enigmatic. In a recent publication it has been suggested that these genes may have no function for secondary metabolite biosynthesis. An in-frame deletion of cloY in the clorobiocin cluster has now been carried out. When the modified cluster was expressed in the heterologous host Streptomyces coelicolor M512, clorobiocin was still formed. However, when the two further mbtH-like genes from elsewhere in the host genome were inactivated as well, clorobiocin formation was reduced dramatically. Complementation with cloY or with any of three other mbtH-like genes restored clorobiocin formation. This is the first report proving the requirement of an mbtH-like gene for secondary metabolite formation, and the first proof that different mbtH-like genes can functionally replace each other. Feeding of an mbtH-defective triple mutant strain with an intact 3-amino-4,7-dihydroxy-coumarin moiety restored antibiotic production, showing that cloY is specifically required for the formation of this moiety of the clorobiocin molecule.
AgnoliK.,
LoweC. A.,
FarmerK. L.,
HusnainS. I.,
ThomasM. S.2006; The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon. J Bacteriol 188:3631–3644[CrossRef]
BentleyS. D.,
ChaterK. F.,
Cerdeno-TarragaA. M.,
ChallisG. L.,
ThomsonN. R.,
JamesK. D.,
HarrisD. E.,
QuailM. A.,
KieserH.other authors2002; Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2. Nature 417:141–147[CrossRef]
BlodgettJ. A.,
ZhangJ. K.,
MetcalfW. W.2005; Molecular cloning, sequence analysis, and heterologous expression of the phosphinothricin tripeptide biosynthetic gene cluster from Streptomyces viridochromogenes DSM 40736. Antimicrob Agents Chemother 49:230–240[CrossRef]
CarterR. A.,
WorsleyP. S.,
SawersG.,
ChallisG. L.,
DilworthM. J.,
CarsonK. C.,
LawrenceJ. A.,
WexlerM.,
JohnstonA. W.,
YeomanK. H.2002; The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum : their expression in other genera requires ECF sigma factor RpoI. Mol Microbiol 44:1153–1166[CrossRef]
ChallisG. L.,
RavelJ.2000; Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase. FEMS Microbiol Lett 187:111–114[CrossRef]
ChenH.,
WalshC. T.2001; Coumarin formation in novobiocin biosynthesis: β -hydroxylation of the aminoacyl enzyme tyrosyl- S -NovH by a cytochrome P450 NovI. Chem Biol 8:301–312[CrossRef]
ChenH.,
HubbardB. K.,
O'ConnorS. E.,
WalshC. T.2002; Formation of β -hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chem Biol 9:103–112[CrossRef]
ChiuH.-T.,
HubbardB. K.,
ShahA. N.,
EideJ.,
FredenburgR. A.,
WalshC. T.,
KhoslaC.2001; Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster. Proc Natl Acad Sci U S A 98:8548–8553[CrossRef]
DatsenkoK. A.,
WannerB. L.2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645[CrossRef]
DoumithM.,
WeingartenP.,
WehmeierU. F.,
Salah-BeyK.,
BenhamouB.,
CapdevilaC.,
MichelJ.-M.,
PiepersbergW.,
RaynalM.-C.2000; Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea. Mol Gen Genet 264:477–485[CrossRef]
EdwardsD. J.,
GerwickW. H.2004; Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J Am Chem Soc 126:11432–11433[CrossRef]
Eustáquio,A. S.,
GustB.,
LuftT.,
LiS.-M.,
ChaterK. F.,
HeideL.2003a; Clorobiocin biosynthesis in Streptomyces . Identification of the halogenase and generation of structural analogs. Chem Biol 10:279–288[CrossRef]
Eustáquio, A. S.,
LuftT.,
WangZ.-X.,
GustB.,
ChaterK. F.,
LiS.-M.,
HeideL.2003b; Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch Microbiol 180:25–32[CrossRef]
Eustáquio, A. S.,
LiS.-M.,
HeideL.2005b; NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis. Microbiology 151:1949–1961[CrossRef]
FlatmanR. H.,
HowellsA. J.,
HeideL.,
FiedlerH. P.,
MaxwellA.2005; Simocyclinone D8, an inhibitor of DNA gyrase with a novel mode of action. Antimicrob Agents Chemother 49:1093–1100[CrossRef]
FlatmanR. H.,
EustaquioA.,
LiS. M.,
HeideL.,
MaxwellA.2006; Structure–activity relationships of aminocoumarin-type gyrase and topoisomerase IV inhibitors obtained by combinatorial biosynthesis. Antimicrob Agents Chemother 50:1136–1142[CrossRef]
FreitagA.,
GalmU.,
LiS.-M.,
HeideL.2004; New aminocoumarin antibiotics from a cloQ -defective mutant of the clorobiocin producer Streptomyces roseochromogenes DS12.976. J Antibiot 57:205–209[CrossRef]
FreitagA.,
WemakorE.,
LiS.-M.,
HeideL.2005; Acyl transfer in clorobiocin biosynthesis: involvement of several proteins in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar. Chembiochem 6:2316–2325[CrossRef]
GarneauS.,
DorresteinP. C.,
KelleherN. L.,
WalshC. T.2005; Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis. Biochemistry 44:2770–2780[CrossRef]
GunsiorM.,
BreazealeS. D.,
LindA. J.,
RavelJ.,
JancJ. W.,
TownsendC. A.2004; The biosynthetic gene cluster for a monocyclic beta-lactam antibiotic, nocardicin A. Chem Biol 11:927–938[CrossRef]
GustB.,
ChallisG. L.,
FowlerK.,
KieserT.,
ChaterK. F.2003; PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100:1541–1546[CrossRef]
LiS.-M.,
HeideL.2004; Functional analysis of biosynthetic genes of aminocoumarins and production of hybrid antibiotics. Curr Med Chem Anti-Infect Agents 3:279–295[CrossRef]
LomboF.,
VelascoA.,
CastroA.,
de la CalleF.,
BranaA. F.,
Sanchez-PuellesJ. M.,
MendezC.,
SalasJ. A.2006; Deciphering the biosynthesis pathway of the antitumor thiocoraline from a marine actinomycete and its expression in two Streptomyces species. Chembiochem 7:366–376[CrossRef]
PacholecM.,
HillsonN. J.,
WalshC. T.2005; NovJ/NovK catalyze benzylic oxidation of a β -hydroxyl tyrosyl- S -pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis. Biochemistry 44:12819–12826[CrossRef]
PagetM. S.,
ChamberlinL.,
AtrihA.,
FosterS. J.,
ButtnerM. J.1999; Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 181:204–211
PojerF.,
LiS.-M.,
HeideL.2002; Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology 148:3901–3911
QuadriL. E.,
SelloJ.,
KeatingT. A.,
WeinrebP. H.,
WalshC. T.1998; Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem Biol 5:631–645[CrossRef]
RedenbachM.,
KieserH. M.,
DenapaiteD.,
EichnerA.,
CullumJ.,
KinashiH.,
HopwoodD. A.1996; A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21:77–96[CrossRef]
SosioM.,
StinchiS.,
BeltramettiF.,
LazzariniA.,
DonadioS.2003; The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by Nonomuraea species. Chem Biol 10:541–549[CrossRef]
StegmannE.,
RauschC.,
StockertS.,
BurkertD.,
WohllebenW.2006; The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production. FEMS Microbiol Lett 262:85–92[CrossRef]
WangG.,
NieL.,
TanH.2003; Cloning and characterization of sanO , a gene involved in nikkomycin biosynthesis in Streptomyces ansochromogenes. Lett Appl Microbiol 37:452–457[CrossRef]
XuH.,
KahlichR.,
KammererB.,
HeideL.,
LiS.-M.2003; CloN2, a novel acyltransferase involved in the attachment of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin. Microbiology 149:2183–2191[CrossRef]
YeatsC.,
BentleyS.,
BatemanA.2003; New knowledge from old: in silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 3:3[CrossRef]