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

Abstract

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 alpha-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.

Figure 1

Biosynthesis of desferrioxamine B from lysine. The desferrioxamine B component binds Fe³⁺ to form ferrioxamine B. Lysine decarboxylase encoded by the desA gene catalyzes the first step of the biosynthetic pathway.

Figure 2

A Nucleotide sequence of the promoter operator region of the desA gene containing the -10 and -35 boxes; the transcription start point is indicated as +1. The mRNA is indicated by a wavy line and the putative ribosome binding site GGAGG upstream of the first translated codon ATG is underlined. The convergent thick arrows indicate the palindromic sequence of binding of the DmdR protein (operator). The two vertical arrows indicate the DNA fragment used for the gel mobility shift experiments. B Electrophoretic mobility shift assay of the [³²P]labeled desA probe and DmdR protein from S. coelicolor. Lanes 1, labeled free desA probe; 2, labeled desA, mixed with DmdR (note the band shift); 3, labeled desA with excess unlabeled desA; 4, labeled desA with DmdR and anti-DmdR antibodies; 5, labeled desA with DmdR and 2,2'-dipyridyl (200 mM) without Mn²⁺; 6, labeled desA with DmdR and 2,2'-dipyridyl (200 mM). C Same as in B but with DmdR purified from R. fascians. Lanes 1, labeled desA; 2, labeled desA with DmdR; 3, labeled desA with DmdR and excess unlabeled desA.

Figure 3

Physical map of the four vectors used to quantify iron regulation of the desA promoter. pUL99DA and pUL99ADi are multicopy vectors whereas pUL42DA and pUL42Adi are low copy number vectors; amy indicates the promoter-less amy gene; desAp corresponds to the desA promoter; xylE, catechol oxygenase gene; ter, transcriptional terminator; tsr, thiostrepton resistance gene (see Table 1).

Figure 4

Direct test on agar plates of the reporter α-amylase expression ability of the desA promoter using round patches of growth of pUL99DA and pUL42DA transformants in LS medium containing 2,2'-dipyridyl as compared to transformants with pULVD10 in which the reporter amylase gene is under the control of the saf promoter. Control transformants with pUL99ADi and pUL42ADi are shown on the right. The plate was stained with I₂ + IK; the figure is a negative of the plate photograph.

Figure 5

Iron regulation of expression of the reporter α-amylase gene coupled to the desA promoter in a multicopy transformant (pUL99DA) (panel A) and a low-copy number transformant (pUL99DA) (panel B) expressed as volumetric enzyme activity (panels A and B) or as specific activity (panel C). ○, ●, pUL99DA; ▼, ▽, pUL42DA. Closed symbols, control cultures in LS medium; open symbols, cultures induced with the iron-complexing agent 2,2'-dypirydyl added at time 0 (inclined arrows).

Figure 6

Expression of the reporter α-amylase gene from the iron-regulated desA promoter versus the non-regulated saf promoter. Experimental conditions were as in Fig. 5. ○, ●, pUL99DA; ▼, ▽, pULVD10. Closed symbols control cultures in LS medium; open symbols cultures induced by addition of the iron-chelator 2,2'-dipyridyl at time 0 (vertical arrows). Results are given as volumetric enzyme levels (panel A) or as specific enzyme production (panel B). The inclined arrows indicate the time of addition (0 h) of the 2,2'-dipyridyl inducer