Characterization of the iron-regulated desA promoter of Streptomyces pilosus as a system for controlled gene expression in actinomycetes

被引:23
作者
Flores, Francisco J. [1 ]
Rincon, Javier [2 ]
Martin, Juan F. [1 ,2 ]
机构
[1] Univ Leon, Area Microbiol, Fac Ciencias Biol & Ambientales, E-24071 Leon, Spain
[2] Inst Biotechnol INBIOTEC, Leon 24006, Spain
关键词
Streptomyces; Electrophoretic Mobility Shift Assay; Dipyridyl; desA Gene; High Iron Affinity;
D O I
10.1186/1475-2859-2-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: The bioavailability of iron is quite low since it is usually present as insoluble complexes. To solve the bioavailability problem microorganisms have developed highly efficient iron-scavenging systems based on the synthesis of siderophores that have high iron affinity. The systems of iron assimilation in microorganisms are strictly regulated to control the intracellular iron levels since at high concentrations iron is toxic for cells. Streptomyces pilosus synthesizes the siderofore desferrioxamine B. The first step in desferrioxamine biosynthesis is decarboxylation of L-lysine to form cadaverine, a desferrioxamine B precursor. This reaction is catalyzed by the lysine decarboxylase, an enzyme encoded by the desA gene that is repressed by iron. Results: The binding of the DmdR (acronym for divalent metal dependent repressor) to the desA promoter in presence of Fe2+ or other divalent ions has been characterized. A 51 bp DNA fragment of the desA promoter containing the 9 bp inverted repeat was sufficient for binding of the DmdR repressor, as observed by the electrophoretic mobility shift assay. The desA mobility shift was prevented by neutralizing DmdR with anti-DmdR antibodies or by chelating the divalent metal in the binding reaction with 2,2'-dipyridyl. Binding to the desA promoter was observed with purified DmdR repressors of Streptomyces coelicolor or Rhodococcus fascians suggesting that there is a common mechanism of iron-regulation in actinomycetes. The complete desA promoter region was coupled using transcriptional fusions to the amy reporter gene (encoding a-amylase) in low copy or multicopy Streptomyces vectors. The iron-regulated desA promoter was induced by addition of the iron chelating agent 2,2'-dipyridyl resulting in a strong expression of the reporter gene. Conclusions: The iron-regulated desA promoter can be used for inducible expression of genes in Streptomyces species, as shown by de-repression of the promoter when coupled to a reporter gene.
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页数:10
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共 36 条
[1]  
[Anonymous], 1987, Besthesda Res Lab Focus
[2]   Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains [J].
Arahou M. ;
Diem H.G. ;
Sasson A. .
World Journal of Microbiology and Biotechnology, 1997, 14 (1) :31-36
[3]   STOFFWECHSELPRODUKTE VON ACTINOMYCETEN .26. UBER DIE ISOLIERUNG UND CHARAKTERISIERUNG DER FERRIOXAMINE A-F, NEUER WUCHSSTOFFE DER SIDERAMIN-GRUPPE [J].
BICKEL, H ;
BOSSHARDT, R ;
GAUMANN, E ;
REUSSER, P ;
VISCHER, E ;
VOSER, W ;
WETTSTEIN, A ;
ZAHNER, H .
HELVETICA CHIMICA ACTA, 1960, 43 (07) :2118-2128
[4]  
Braun V, 1997, BIOL CHEM, V378, P779
[5]  
BRENNAN RG, 1986, CHEM SCRIPTA, V26B, P251
[6]  
Bullen J J, 1978, Curr Top Microbiol Immunol, V80, P1
[7]   amy as a reporter gene for promoter activity in Nocardia lactamdurans: Comparison of promoters of the cephamycin cluster [J].
Chary, VK ;
delaFuente, JL ;
Liras, P ;
Martin, JF .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (08) :2977-2982
[8]  
Chater K. F., 1982, CURR TOP MICROBIOL I, V96, P69
[9]  
Clayton M, 1990, NUCLEIC ACIDS RES, V18, P1077