Dual control system - A novel scaffolding architecture of an inducible regulatory device for the precise regulation of gene expression.

Here, we present a novel scaffolding architecture of an inducible regulatory device. This dual control system is completely silent in the off stage and is coupled to the regulation of gene expression at both the transcriptional and translational levels. This system also functions as an AND gate. We demonstrated the effectiveness of the cumate-riboswitch dual control system for the control of pamamycin production in Streptomyces albus. Placing the cre recombinase gene under the control of this system permitted the construction of synthetic devices with non-volatile memory that sense the signal and respond by altering DNA at the chromosomal level, thereby producing changes that are heritable. In addition, we present a library of synthetic inducible promoters based on the previously described cumate switch. With only one inducer and different promoters, we demonstrate that simultaneous modulation of the expression of several genes to different levels in various operons is possible. Because all modules of the AND gates are functional in bacteria other than Streptomyces, we anticipate that these regulatory devices can be used to control gene expression in other Actinobacteria. The features described in this study make these systems promising tools for metabolic engineering and biotechnology in Actinobacteria.

Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is the producer of phosphoglycolipid antibiotics moenomycins that for almost 40 years were used worldwide as an animal feed additive. As the use of moenomycins narrows down (due to bans in the EU and some other countries), it opens the opportunity to develop much-needed antibiotics against Gram-positive human pathogens, such as cocci. It is desirable to develop ATCC14672 strains accumulating only certain members of moenomycin family which would facilitate their purification, analysis and/or chemical modification. Here we tested site-specific recombinases (SSRs) as a tool to manipulate the genome of ATCC14672 and to achieve aforementioned goals. We show that of three SSRs tested--Cre, Dre, and Flp--the first two efficiently catalyzed recombination reactions, while Flp showed no activity in ATCC14672 cells. Cre recombinase can be reused at least three times to modify ATCC14672 genome without detrimental effects, such as large-scale inversions or deletions. Properties of the generated strains and SSRs are discussed.

Generation and study of the strains of streptomycetes - heterologous hosts for production of moenomycin.

Moenomycins (Mm) are family of phosphoglycolipid natural products that is considered a blueprint to develop new class of antibiotics. The natural Mm producer, Streptomyces ghanaensis (ATCC14672), produces very low amounts of moenomycin, fueling the investigations on genetic approaches to improve its titers. Heterologous expression of moenomycin biosynthesis gene cluster (moe) would be one of the ways to reach this goal. Here we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family), and determine their potential for the antibiotic production. The rpoB point mutation in the model strain of Streptomyces genetics, S. coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed here for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes.

Complete genome sequence of Streptomyces fulvissimus.

The complete genome sequence of Streptomyces fulvissimus (DSM 40593), consisting of a linear chromosome with a size of 7.9Mbp, is reported. Preliminary data indicates that the chromosome of S. fulvissimus contains 32 putative gene clusters involved in the biosynthesis of secondary metabolites, two of them showing very high similarity to the valinomycin and nonactin biosynthetic clusters. The availability of genome sequence of S. fulvissimus will contribute to the evaluation of the full biosynthetical potential of streptomycetes.

Surprising production of a new urdamycin derivative by S. fradiae Delta urdQ/R.

A strain (S. fradiae Delta urdQ/R) with mutations in urdQ and urdR encoding a dTDP-hexose-3,4-dehydratase and a dTDP-hexose-4-ketoreductase, respectively, produces a new urdamycin analogue (urdamycin X) with changes in the polyketide structure. The structure of urdamycin X has been elucidated by NMR spectroscopy. Urdamycin X was not detectable, even in small amounts, in either S. fradiae Delta urdQ, in S. fradiae DeltaurdR or in S. fradiae A0, a mutant lacking all glycosyltransferase genes. Complementation of S. fradiae Delta urdQ/R restored urdamycin A production indicating that the mutations did not cause any polar effect.

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes.

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.

DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912.

The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912. LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure. Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE. Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription. Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass. In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.

Targeted disruption of Streptomyces globisporus lndF and lndL cyclase genes involved in landomycin E biosynthesis.

Streptomyces globisporus strains with knockouts in lndF and lndL genes, previously identified as possibly encoding cyclases governing cyclization of the nascent oligoketide ('polyketide') chain during the biosynthesis of the antitumor angucycline landomycin E, were prepared. On combining the results of sequence analysis and HPLC of extracts from mutant strains, lndL was suggested to control the first cyclization-aromatization event and lndF to be responsible for the 3rd-4th ring formation.

Production of landomycins in Streptomyces globisporus 1912 and S cyanogenus S136 is regulated by genes encoding putative transcriptional activators.

The regulatory genes lanI and lndI have been cloned from the landomycin A (LaA) producer Streptomyces cyanogenus S136 and from the landomycin E (LaE) producer Streptomyces globisporus 1912, respectively and both have been sequenced. A culture of S. globisporus I2-1 carrying a disrupted lndI gene did not produce LaE and other related intermediates. Complementation of S. globisporus I2-1 with either the lndI or lanI gene reconstituted LaE production indicating that LanI and LndI are involved in activation of structural genes in the respective clusters. Structural features of these regulatory genes are discussed.