1 Department of Genetics and Biotechnology of Ivan Franko National University of L'viv, Grushevskogo st.4, L'viv 79005, Ukraine
2 Department of Microbiology, Niigata University of Pharmacy, Kami-Shinei-cho 5-13-2, Niigata 950-2081, Japan
3 Institut für Pharmazeutische Wissenschaften, Lehrstuhl für Pharmazeutische Biologie und Biotechnologie Albert-Ludwigs-Universität Freiburg, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany
4 Department of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, Kami-Shinei-cho 5-13-2, Niigata 950-2081, Japan
Correspondence
V. Fedorenko
v_fedorenko{at}franko.lviv.ua
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY662671.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The focus of this research is Streptomyces globisporus 1912, the producer of the potent antitumour drug landomycin E (LaE) (Fig. 1a). It shows an interesting spectrum of antitumour activities (Krohn & Rohr, 1997
; Polishchuk et al., 1996
). The LaE biosynthetic gene cluster (lnd) has been cloned (Fedorenko et al., 2000
) and the gene lndI, encoding a putative transcriptional activator responsible for the activation of all other lnd genes, has been identified via gene disruption and complementation studies (Pankevych et al., 2001
; Rebets et al., 2003
).
|
A gene very similar to lndI has been identified in the landomycin A biosynthetic gene cluster of Streptomyces cyanogenus S136. Both genes have been proven to be functionally interchangeable (Rebets et al., 2003). Interestingly these proteins contain a set of different amino acids in the
-loops of their winged-helix domains, which are thought to be crucial for DNA recognition and RNA polymerase recruitment (Wietzorreck & Bibb, 1997
). These differences might cause a higher level of landomycin production by S. cyanogenus S136 (Rebets et al., 2003
). Detailed studies of the mechanisms of transcriptional activation of secondary metabolism pathways will help (i) to elucidate the complex interaction of regulatory proteins during physiological differentiation of Streptomyces, (ii) the understanding of the regulation of production of angucycline antibiotics and (iii) the development of rational approaches towards the generation of a LaE overproducer.
Here we report results of experiments supporting our theory of LndI as an autoregulatory protein that binds to a promoter region of its own gene lndI and to promoter regions of structural lnd genes thus triggering LaE biosynthesis. Using the enhanced green fluorescent protein (EGFP) reporter system we have studied the temporal and spatial expression profile of the regulatory gene lndI.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasmid construction
(i) For lndI expression in Streptomyces.
For simultaneous expression of the lndI gene and the EGFP gene fused to the lnd promoter regions, pKC1218Ea was generated. The spectinomycin-resistance (Spr) gene cassette aadA was excised from pHP45 (Prentki & Krisch, 1984
) as a HindIII fragment. It was treated with the Klenow fragment and cloned into the blunt-ended XhoI site of pKC1218E (Rodriguez et al., 2002
), which is located within the apramycin-resistance gene aac(3)IV. For the generation of pKCEaI lndI was cloned on a 3 kbp EcoRI fragment (Fig. 1b
) into pKC1218Ea.
(ii) For in vivo titration of LndI.
Putative promoter sequences were searched with GENETYX-MAC8.1 and DNASTAR software. Promoter regions of the lndI (PlndI) and lndE (PlndE) genes were subcloned from pSI2-9 as HindIIIHindIII and EcoRVEcoRI fragments (Fig. 1b; second HindIII site, adjacent to EcoRI at the end of lndI, comes from polylinker of pSI2-9), respectively, into pBluescriptIISK to give pBLH (containing PlndI) and pBLE (containing PlndE). The GenBank accession number for lndI and its promoter region sequence is AY659998. The sequence of the lndE upstream region has been submitted to GenBank under accession number AY662671. To generate pSOH PlndI was retrieved from pBLH as an EcoRIBamHI fragment and cloned into pSOK101. To generate pSOE an EcoRVEcoRI fragment from pBLE containing PlndE was subcloned into EcoRVEcoRI-digested pSOK101 (Fig. 1b
).
(iii) For expression of LndI as a CBD-fusion protein.
For LndI expression using the pTYB2 IMPACT-CN system, primers Lnde1 and Lec1Rev (5'-ATACATATGAGACGGCAGTCTGCA and 5'-AATGATATCAGGAGATCCGAGCCGGAA, respectively; NdeI and EcoRV sites underlined) were used to amplify lndI. The PCR product was cloned into pT7Blue T-vector to generate pT7BluelndI-2. The lndI gene was excised from this plasmid as an NdeIEcoRV fragment and cloned into the NdeISmaI-digested vector pTYB2 to fuse lndI with the intein-chitin-binding domain (CBD) coding region. The generated plasmid was named pSIZT4.
(iv) For studying transcriptional activity of lnd promoters.
The EcoRVKpnI fragment carrying PlndI was excised from pBLH and was ligated into the respective sites of pIJ8660 to generate a transcriptional unit with PlndI and the EGFP gene. The plasmid was named pIJ8660H. To shorten the lndI gene promoter region to a final length of 270 bp an NdeI fragment was cloned from pIJ8660H into the respective site of pIJ8660 to create pIJ8660N. PlndE was subcloned as an EcoRVBamHI fragment from pBLE into pIJ8660 to generate pIJ8660E.
Overexpression and purification of LndI.
E. coli BL21(DE3) strains harbouring plasmids for LndI expression were grown in TB at 36 °C to an optical density at 600 nm of 0·5, induced with IPTG (0·4 mM) and incubated for an additional 17 h at 16 °C. His-tagged LndI purification was performed essentially as recommended by commercial suppliers for native conditions (Novagen).
Cells of E. coli BL21(DE3) harbouring pSIZT4 were resuspended in binding buffer [10 mM Tris/HCl (pH 7·5), 500 mM NaCl, 0·5 mM EDTA]. After disruption by sonication, proteins were loaded onto a chitin-agarose column (NEB) and washed with 10 vols of binding buffer. CBD-fusion-protein purification was performed as directed by the manufacturer (NEB).
Protein analysis.
Protein concentrations were determined by the BCA (bicinchoninic acid) protein assay (PIERCE) with bovine serum albumin (BSA) as standard. SDS-PAGE was performed according to the method of Laemmli (Sambrook & Russell, 2001), and the gels were Coomassie-blue-R-stained. For total protein analysis the cells were suspended in SDS-PAGE sample buffer [50 mM Tris/HCl (pH 6·8), 100 mM DTT, 2 % SDS (w/v), 0·1 % bromophenol blue (w/v), and 10 % glycerol (v/v)] and heated in boiling water for 7 min. After centrifugation the supernatant was run on an SDS-12 % polyacrylamide gel.
Growth dynamics of S. globisporus strains were studied by measuring the total protein concentration in the samples taken from culture broth each 12 h of growth. A 1 ml sample of liquid culture was washed twice with deionized water and resuspended in 10 % trichloroacetic acid for 60 min at 37 °C. Cells were centrifuged and incubated in 0·4 M NaOH for 12 h.
DNAprotein-binding assays.
A 467 bp EcoRIHindIII fragment from pBLH (PlndI), a 557 bp EcoRIEcoRV fragment from pBLE (PlndE) and a 313 bp KpnIPstI fragment from pBlue1.8K (Fig. 1b) were used in the assays. The GenBank accession number of the sequence for lndF and the surrounding regions is AY659997. All fragments were labelled from both ends with fluorescein using a 5-IAF-labelling kit as described by the supplier (Amersham Biosciences). DNA-binding tests were performed by the electrophoretic mobility-shift assay (EMSA) (Taylor et al., 1994
) with 0·5 to 0·7 µg of LndI preparation. Fluorescein-labelled DNA (25 ng) was then added, and the reaction mixture was incubated for 20 min at 27 °C. The gels were analysed by scanning with a Typhoon 9400 Variable Mode Imager (Amersham Biosciences). In competition experiments the assay was performed with various amounts of unlabelled competitor DNA.
LaE production analysis.
A pure sample of LaE, kindly provided by Professor J. Rohr (College of Pharmacy, University of Kentucky, KY, USA), was used as an HPLC standard. LaE production was analysed via HPLC as described (Rebets et al., 2003). The levels of LaE production in our experiments are related to equal proportions of total protein in different strains. Measurements were taken in triplicate and the means of the data were found.
Sample preparation and confocal microscopy.
For EGFP-production analysis, mycelia grown in TSB media were washed with deionized water and applied to glass slides. To study cell viability, propidium iodide (PI) was used as an indicator in a procedure described elsewhere (Khetan et al., 2000; Kyung et al., 2001
).
For solid-phase observations, single colonies were grown on thin MM plates (Kieser et al., 2000) containing mannitol instead of glucose, and thin longitudinal slices were removed with a sharp blade and covered with water.
A Fluoroview confocal system (Olympus) with an Olympus OL BX50 microscope and an argon laser (providing excitation at 488 nm) was used to observe S. globisporus strains with GFP expression and PI staining. FITC- (emission at 506535 nm) and PI- (emission at 600615 nm) filter sets were used to observe green and red fluorescence, respectively. Green fluorescence and transition or PI images were obtained simultaneously by using separate detectors. To gain high reliability in quantitative analysis of captured images the same operation parameters were used for samples at the same time point. The confocal images were saved as TIFF files and image analysis was performed by FLUOROVIEW 2.1 software (Olympus). EGFP expression was quantified by measuring the fluorescence intensity within a rectangle on each image (Kataoka et al., 1999).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To prove that LndI is a central regulatory protein in vivo LndI titration experiments were undertaken. PlndI and PlndE were chosen for these studies. pIJ101-based plasmids pSOH and pSOE, containing PlndI and PlndE, respectively, were introduced into S. globisporus 1912. LaE production was significantly reduced in both pSOH- (to 71 % of the wild-type level) and pSOE- (to 43 % of the wild-type level) harbouring strains. This effect can be explained by titration of LndI by the plasmid-borne PlndI and PlndE. The negative effect on LaE production was more pronounced in strain 1912(pSOE). More efficient competition of PlndE for the LndI protein possibly reflects higher affinity of LndI for the lndE promoter than for the promoter of its own gene. From these results we can also conclude indirectly that LndI binds to its own promoter region.
LndI was overexpressed in E. coli and purified as an intein-CBD-tagged protein
All attempts to purify sufficient quantities of soluble 6-His-tagged LndI were unsuccessful. The level of recombinant LndI production was low and, under conditions stated, less than half of the protein was present in soluble fraction (confirmed by SDS-PAGE; data not shown). To avoid the problems of low protein solubility we pursued a strategy to clone and express LndI as a C-terminal CBD-fusion protein. Using LndIinteinCBD fusion, we obtained highly soluble LndI, which was easily purified by a one-step column procedure. A high concentration of recombinant LndI protein with an additional asparagine residue in the C-terminal part was obtained after intein cleavage (Fig. 2).
|
|
Results of in vitro analysis of LndI-binding activity are in good agreement with the LndI in vivo titration experiment. We assume LndI to be a DNA-binding regulatory protein that controls transcription of the structural lnd genes and its own lndI gene.
Transcriptional fusion of the lndI promoter region to the EGFP reporter gene allows analysis of temporal and spatial lndI expression
EGFP has been shown to be a convenient reporter system in studies on the temporal and spatial expression of various streptomycete genes (Sun et al., 1999). EGFP expression levels can be quantified reliably (Kataoka et al., 1999
), and its use does not cause any artifacts that would result from a highly stable reporter protein (Khetan et al., 2000
; Kyung et al., 2001
). To analyse the spatial and temporal expression pattern of the lndI gene the
C31-based plasmid pIJ8660H, carrying PlndI transcriptionally fused to the EGFP gene, was constructed. pIJ8660H was introduced into S. globisporus 1912. Integration was confirmed by Southern blot analysis (data not shown). Spore suspensions of strains 1912(pIJ8660H) and 1912(pIJ8660) were inoculated into 250 ml baffled flasks with 50 ml TSB, and samples of the strains were taken at different culture times for confocal scanning microscopy, and total protein and LaE production determination. Results of these experiments are presented in Fig. 4
and summarized in Fig. 5
.
|
|
In the batch culture of S. globisporus 1912(pIJ8660H) no fluorescence was detectable during the first 12 h of growth. After 12 h slight EGFP production was detectable in culture samples. It was much higher after 24 h, with a maximum after 48 to 60 h. After 60 h the intensity of the fluorescence decreased strongly. LaE synthesis was not detectable during the first 12 h. After 24 h LaE was detectable, reaching peak production level after 48 to 60 h (Fig. 5).
To examine whether the decrease in EGFP as well as LaE production was caused by cell autolysis, cell staining with PI was performed. After 72 h the intensity of green fluorescence decreased while red fluorescence significantly increased, and almost all of the mycelia exhibited red fluorescence, indicating the death of the cells (Fig. 6). During this time increasing decay of mycelia was observed. However, even after 96 h weak green fluorescence was observed, suggesting that lndI was still expressed in old mycelia.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently several gene clusters for angucycline antibiotics have been cloned and sequenced. Genes encoding putative transcriptional activators were identified within these clusters (Fedorenko et al., 2000; Trefzer et al., 2002
; Westrich et al., 1999
; Yang et al., 2001
). They are distinct from other members of the SARP family in that they contain a winged helixturnhelix DNA-binding domain in their C-terminal region (Wietzorreck & Bibb, 1997
). Promoter regions of angucycline biosynthetic genes appear to not contain typical SARP-like target DNA sequences, suggesting a different mechanism for controlling the production of these compounds.
LaE is a highly cytotoxic angucycline showing also moderate antibacterial activity (Krohn & Rohr, 1997; Polishchuk et al., 1996
). It is produced at a low level in the wild-type strain. As a potentially toxic compound, LaE production should be under strict control to avoid its undesired overproduction. LndI has been identified as a putative transcriptional activator of the LaE cluster. Interestingly lndI contains the rare TTA codon that might limit lndI expression (Pankevych et al., 2001
) and is a characteristic for other antibiotic biosynthesis regulators. Our data show that, besides temporal regulation, lndI expression is also the subject of spatial control as is evident from the analysis of PlndI-driven EGFP expression in S. globisporus colonies grown on solid medium.
Based on in vivo LndI titration experiments, gel mobility-shift assays and promoter-probing studies LndI can be classified as an autoregulatory DNA-binding protein. To our knowledge, this is the first research work on an autoregulatory pathway-specific transcriptional factor involved in polyketide biosynthesis in actinomycetes. A similar function has been described for CcaR, a regulatory protein involved in the activation of the -lactam supercluster (Santamarta et al., 2002
). However, the extent of lndI gene activation by LndI is much lower than that of lndE. Thus the biological significance of lndI autoregulation should be addressed in additional experiments. We showed here that LndI binds to the lndE promoter region and to the lndI promoter, and that direct repeats tandemly localized upstream of lndI are not involved in lndI regulation. However, additional biochemical experiments are still required to understand the DNA-binding features of LndI.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bourn, W. R. & Babb, B. (1995). Computer assisted identification and classification of streptomycete promoters. Nucleic Acids Res 23, 36963703.[Abstract]
Chater, K. F. (1993). Genetics of differentiation in Streptomyces. Annu Rev Microbiol 47, 685713.[CrossRef][Medline]
Fedorenko, V., Basiliya, L., Pankevych, K., Dubitska, L., Ostash, B., Luzhetskyy, A., Gromyko, O. & Krugel, H. (2000). Genetic control of antitumor antibiotics-polyketides by actinomycetes. Bull Inst Agr Microbiol 8, 2731 (in Ukrainian).
Fernandez-Moreno, M. A., Caballero, J. L., Hopwood, D. A. & Malpartida, F. (1991). The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell 66, 769780.[Medline]
Kataoka, M., Kosono, S. & Tsujimoto, G. (1999). Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony. FEBS Lett 462, 425429.[CrossRef][Medline]
Khetan, A., Hu, W.-S. & Sherman, D. H. (2000). Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter. Microbiology 146, 18691880.[Medline]
Kieser, T., Bibb, M. J., Buttner, J. M., Chater, K. F. & Hopwood, D. A. (2000). Practical Streptomyces Genetics. Norwich, UK: John Innes Foundation.
Krohn, K. & Rohr, J. (1997). Angucyclines: total syntheses, new structures and biosynthetic studies of an emerging new class of antibiotics. Top Curr Chem 188, 127195.
Kyung, Y.-S., Hu, W.-S. & Sherman, D. H. (2001). Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein. Mol Microbiol 40, 530541.[CrossRef][Medline]
Luzhetskii, A. N., Ostash, B. E. & Fedorenko, V. A. (2001). Intergeneric conjugation Escherichia coli - Streptomyces globisporus 1912 with using of integrative plasmid pSET152 and its derivative. Genetika 37, 13401347 (in Russian).[Medline]
Martinez-Hackert, E. & Stock, A. M. (1997). The DNA-binding domain of OmpR: crystal structure of a winged helix transcription factor. Structure 5, 109124.[Medline]
Narva, K. E. & Feitelson, J. S. (1990). Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2). J Bacteriol 172, 326333.[Medline]
Ostash, B., Rebets, Yu., Yuskevich, V., Luzhetskyy, A., Tkachenko, V. & Fedorenko, V. (2003a). Targeted disruption of Streptomyces globisporus lndF and lndL cyclase genes involved in landomycin E biosynthesis. Folia Microbiol 48, 484488.
Ostash, B., Rebets, Yu., Samborskyy, M., Salas, J. A. & Fedorenko, V. (2003b). Sequencing and analysis of putative 3-d and 4-th ring cyclase gene lndF of Streptomyces globisporus 1912 landomycin E biosynthesis gene cluster. Visn L'viv Univ Ser Biol 32, 8491.
Pankevych, K., Kruegel, H. & Fedorenko, V. (2001). Cloning and sequencing of a putative positive transcription regulator gene of landomycin E biosynthetic gene cluster of Streptomyces globisporus 1912. Visn L'viv Univ Ser Biol 27, 97105.
Paradkar, A. S., Aidoo, K. A. & Jensen, S. E. (1998). A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus. Mol Microbiol 27, 831843.[CrossRef][Medline]
Polishchuk, L. V., Hanusevych, I. I. & Matseliukh, B. P. (1996). The antitumor action of antibiotics produced by Streptomyces globisporus 1912 studied in a model of Guerin's carcinoma in rats. Mikrobiol Zhurn 58, 5558 (in Ukrainian).
Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303313.[CrossRef][Medline]
Rebets, Y., Ostash, B., Luzhetskyy, A., Hoffmeister, D., Brana, A., Mendez, C., Salas, J. A., Bechthold, A. & Fedorenko, V. (2003). Production of landomycins in Streptomyces globisporus 1912 and S. cyanogenus S136 is regulated by genes encoding putative transcriptional activators. FEMS Microbiol Lett 222, 149153.[CrossRef][Medline]
Rodriguez, L., Aguirrezabalaga, I., Allende, N., Brana, A., Mendez, C. & Salas, J. A. (2002). Engineering deoxysugar biosynthetic pathways from antibiotic-producing microorganisms. A tool to produce novel glycosylated bioactive compounds. Chem Biol 9, 721729.[CrossRef][Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Santamarta, I., Rodríguez-García, A., Pérez-Redondo, R., Martín, J. F. & Liras, P. (2002). CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus. J Bacteriol 184, 31063113.
Stephanopoulos, G. (2002). Metabolic engineering by genome shuffling. Nat Biotechnol 20, 666668.[CrossRef][Medline]
Stutzman-Engwall, K. J., Otten, S. L. & Hutchinson, C. R. (1992). Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol 174, 144154.[Abstract]
Sun, J., Kelemen, G. H., Fernandez-Abalos, J. M. & Bibb, M. J. (1999). Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2). Microbiology 145, 22212227.[Medline]
Tang, L., Grimm, A., Zhang, Y.-X. & Hutchinson, C. R. (1996). Purification and characterization of the DNA-binding protein DnRI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius. Mol Microbiol 22, 801813.[CrossRef][Medline]
Taylor, J. D., Ackroyd, A. J. & Halford, S. E. (1994). The gel shift assay for the analysis of DNAprotein interaction. In Methods in Molecular Biology. DNAProtein Interactions, pp. 263281. Edited by G. G. Kneale. Totowa, NJ: Humana Press.
Trefzer, A., Pelzer, S., Schimana, J., Stockert, S., Bihlmaier, C., Fiedler, H. P., Welzel, K., Vente, A. & Bechthold, A. (2002). Biosynthetic gene cluster of simocyclinone, a natural multihybrid antibiotic. Antimicrob Agents Chemother 46, 11741182.
Westrich, L., Domann, S., Faust, B., Bedford, D., Hopwood, D. A. & Bechthold, A. (1999). Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin A biosynthesis. FEMS Microbiol Lett 170, 381387.[CrossRef][Medline]
Wietzorreck, A. & Bibb, M. (1997). A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA binding fold. Mol Microbiol 25, 11811184.[CrossRef][Medline]
Yang, K., Han, L., He, J., Wang, L. & Vining, L. C. (2001). A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230. Gene 279, 165173.[CrossRef][Medline]
Zotchev, S., Haugan, K., Sekurova, O., Sletta, H., Ellingsen, T. E. & Valla, S. (2000). Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455. Microbiology 146, 611619.[Medline]
Received 15 April 2004;
revised 6 September 2004;
accepted 27 September 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |