Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

Abstract

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any 'genetic toolkit' is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1-100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1-1 microg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer.

Figure 1

Regulation of synthetic tcps by aTc using neo as a reporter gene. (A) Plasmid constructs. All the plasmids are depicted as they would be when integrated into the Streptomyces chromosome φC31 attB site. The genes and elements required for replication, integration and transfer are shown in grey and include the φC31 integrase gene (int), the E.coli replication region (rep), the apramycin resistance gene (aac(3)IV), the origin of transfer (oriT), attL and attR. The tetRiS gene and the neo gene are shown in red and blue, respectively. The synthetic tcps [for their sequences see (B)] are shown as green arrowheads, and the two terminators, t_mmr and t_fd, are shown as shaded pink boxes. pPC700, pPC808, pPC830, pPC840, pPC850 and pPC861 differ only by the sequence of the tcps and contain tcp700, tcp808, tcp830, tcp840, tcp850, tcp861, respectively (B) pAR840 contains no tcp. pPC706, pPS808, pPS830, pPS840, pPS850, pPS861 and pAR850 are derivatives of pPC700, pPC808, pPC830, pPC840 pPC850, pPC861 or pAR840, respectively, that lack the tetRiS gene. (B) Sequences of the tcps are shown. The −10 and −35 promoter elements previously characterized for the ermEp1 promoter are indicated (19). The arrow represents the transcription start (19). Two versions of tcp808 and tcp840 were made, which contain either A (tcp808A, tcp840A) or T (tcp808T, tcp840T) at the last position of the upstream tetO (position −15). The Tn10 tetO1 and tetO2 sequences are shown for information, and the tetO-like elements are shown in bold. There are several differences between the tetO1-like sequences in tcp830 and tcp700 compared with the Tn10 tetO1, where the base pairs from the ermEp1 promoter were maintained. This was performed because the ermEp1 promoter is thought to be of the extended −10 type and changes in this region might severely affect promoter activity (19). (C) Titration of kanamycin resistances conferred by S.coelicolor J1929 strains containing the tcp-neo fusions. The strains were constructed using the plasmids described in (A) and are labelled according to which tcp is driving neo. SMMS agar plates containing increasing amounts of kanamycin and supplemented with either 0 μg/ml aTc or 1.5 μg/ml aTc were inoculated with 5 × 10³ spores suspended in 10 μl of water. The plates were incubated at 30°C and photographed after 67 h.

Figure 2

Regulation of luxAB expression by tcp830 and the innate Tc-inducible promoter, itcp0252. (A and B) Plasmid organization of the tcp830–luxAB fusion (A) and the itcp0252–luxAB fusion (B). pAR933a encodes tcp830 reading towards the luxAB genes and contains an rbs inserted upstream of the start codon for luxA to optimize expression. pAR933b is similar to pAR933a, except that the fragment containing t_mmr, tcp830, luxAB is inverted compared with pAR933a. pAR870 is similar to pAR933a, except that it is ΔtetRiS and lacks the rbs upstream of luxAB. pAR911a and pAR911b encode the innate promoter–operator, itcp0252, and repressor, SCO0253. They are different only in the orientations of the fragment containing t_mmr, SCO0253, itcp0252, luxAB. pAR913a and pAR913b differ with pAR911a and pAR911b, respectively, as they lack the SCO0253 repressor gene. (C) Sequence of itcp0252 is compared with the Tn10 tetO1 and tetO2 sequences. (D and E) Regulation of luciferase driven by tcp830 in S.coelicolor pAR933a (D) and itcp0252 in S.coelicolor pAR911b (E) during a growth course. The black filled symbols and the open symbols indicate the levels of luciferase expression in induced cultures with 1 μg/ml aTc, and in uninduced cultures (0 μg/ml aTc), respectively. The data [277 measurements for both null and induced conditions in (D), and 206 and 187 for the null and induced conditions, respectively, in (E)] were grouped by growth intervals and the means of these and 95% CIs are shown. (F) The IF versus growth was calculated using the data from (D). Above the curve in (D–F) is a growth phase indicator showing how the OD₄₉₂ values correlate with the rapid growth phase (Growth I), the transition phase (TrI and TrII) and the second growth phase (Growth II). Transition phase was divided into TrI and TrII at the point where the growth curve undergoes an inflection.

Figure 3

IF increases with increasing inducer. SLA of cultures grown to between OD₄₉₂ values of 0.64–0.85 for each condition (mean and CIs for at least 46 values) plotted against aTc concentration. The line is a non-linear regression calculated from log₂(IF) = 3.9885 + 4.4295^*{1−exp[−0.4294^* log₂(C)]}, R² = 0.9991, where C is the aTc concentration in ng/ml.

Figure 4

Growth of S.coelicolor J1929 in the presence of different tetracyclines. OD₄₉₂s of each culture were measured over a period of 50 h. The dots represent the mean growth curves of each condition obtained after synchronization of the replicate cultures to the time point where OD₄₉₂ = 0.04, as this is the OD when the linear growth begins for nearly all growth curves; the mean OD₄₉₂ at each time point was then calculated, and the values plotted against time. The lines are the averaged regression functions calculated from the linear ranges of each of 48 replicates (two biological replicates). The slope of the mean regression line gave the average growth rate (μ_c), and the extrapolation of the line to where it crossed the x-axis (an OD of 0.005) gave the arbitrary mean lag time (λ_c). μ_c and λ_c for each condition are shown in Table 3. The mean regression lines and the mean growth curves are colour coded as follows: black, cultures with no addition of inducer; yellow, 0.1 μg/ml aTc; light blue, 0.1 μg/ml Tc; brown, 1 μg/ml aTc; dark blue, 1 μg/ml Tc; green, 1 μg/ml Dc.