Departamento de Biología Molecular y Biotecnología del Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, DF CP 04510, Mexico
Correspondence
Sergio Sánchez
sersan{at}servidor.unam.mx
Elizabeth Langley
langley{at}servidor.unam.mx
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ABSTRACT |
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Present address: Department of Medical Genetics, 8-33 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Present address: Facultad de Medicina, Universidad Autónoma de Campeche, Campeche, Cam. 24090, Mexico.
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INTRODUCTION |
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In various Streptomyces species, glucose exerts CCR that not only affects the utilization of various carbon sources, but also the synthesis of secondary metabolites (Demain, 1989). The mechanisms used by streptomycetes to carry out carbon regulation have been studied primarily in Streptomyces coelicolor. One of these mechanisms is the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS) (Saier et al., 1995
; Stülke & Hillen, 1999
). However, this mechanism responds only to the presence of fructose and N-acetylglucosamine (Nothaft et al., 2003
; Wang et al., 2002
). Thus, in contrast to mechanisms found in enterobacteria, the PTS system appears to play a secondary role in CCR of streptomycetes (Titgemeyer et al., 1995
). Besides the PTS system, the analysis of Streptomyces mutants insensitive to CCR has suggested the involvement of the expression products of a number of genes, such as bld and ccrA (Champness, 1988
; Ingram et al., 1995
). Additionally, Hodgson (1982)
observed that S. coelicolor mutants isolated by their resistance to growth on the glucose analogue 2-deoxyglucose (2-DOG) present a decreased sensitivity to repression by various carbon sources, as well as reduced glucose kinase (Glk) activity. When these 2-DOG-resistant (DogR) mutants were complemented with the glkA gene (Ikeda et al., 1984
), Glk activity was completely restored, while CCR sensitivity was only partially recovered (Angell et al., 1994
), supporting a role for Glk in this regulatory process. However, a complete recovery can be achieved when the 572 bp SCO2127 gene, located just upstream, is introduced with glkA (Angell et al., 1994
). The authors suggest that SCO2127 may encode a protein involved in glucose transport or its metabolism. However, there has been no evidence to support these possibilities.
Our group has worked with DogR mutants from Streptomyces peucetius var. caesius that show a similar phenotype (Segura et al., 1996). However, in addition to presenting CCR insensitivity and low Glk activity, our mutants also show difficulties in transporting glucose (Escalante et al., 1999
), and this phenotype is partially corrected in revertants of a DogR mutant that have recovered 2-DOG sensitivity (DogS). However, although the Glk of these DogS mutants is increased compared with the parent strain, there is no correlation between their Glk activity levels and the degree of glucose repression (Ramos et al., 2004
).
In spite of a large number of studies on the effect of glucose in the genus Streptomyces, the molecular bases for CCR have yet to be clearly established, and there is not a satisfactory regulatory model to explain this phenomenon. With this background, we began to review the effect of the S. coelicolor glkA and SCO2127 genes on Glk activity, glucose transport and CCR sensitivity in a series of mutants derived from S. peucetius var. caesius insensitive to CCR, which present low levels of Glk activity and deficient glucose transport. Our results support a stimulatory role for SCO2127 on CCR in Streptomyces species.
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METHODS |
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Protoplast formation and regeneration.
Protoplast formation was carried out from strains grown in 50 ml YEME medium supplemented with 5 mM MgCl2.6H2O and 0·5 % glycine, as described by Kieser et al. (2000). Protoplast regeneration was carried out in R6 medium (Baltz & Matsushima, 1981
) for 40 h. After regeneration, 1 ml sucrose (10·3 %) and 50 µg thiostrepton were added, and incubation continued for another 48 h.
Southern blot analyses.
The glkA and SCO2127 genes from S. coelicolor were used as probes to detect their possible homologues in DNA from S. peucetius var. caesius. Using the method of Kieser et al. (2000), chromosomal DNA was extracted from 48 h cultures grown in YEME supplemented with 5 mM MgCl2.6H2O. Total DNA from the original S. peucetius var. caesius strain, and from S. coelicolor M145 as a positive control, was digested separately with BamHI, EcoRI, PstI and XhoI. After digestion, 10 µg of each digest was run on a 1 % agarose gel. Southern blotting and hybridization were carried out according to the manufacturer's recommendations for the DIG High-Prime DNA Labelling and Detection Kit II (Roche Molecular Biochemicals).
Anthracycline and protein determination.
Anthracyclines were extracted from harvested mycelia (96 h cultures) using acetone and 0·05 M sulfuric acid (4 : 1), according to Arcamone et al. (1969). Anthracyclines were quantified at 495 nm using a molar absorption coefficient of 220. For protein determination, samples were processed as previously reported (Segura et al., 1997
), and assayed by the Lowry method, using bovine serum albumin as a standard.
Glucose kinase assays.
Glk activity from cell-free extracts prepared from 48 h mycelial cultures of the strains was measured spectrophotometrically by monitoring the reduction of NADP in a glucose-6-phosphate-dehydrogenase-coupled reaction at pH 7·2 and 25 °C, as previously reported (Imriskova et al., 2001; Ramos et al., 2004
). Glk activity was determined at 25 °C and pH 7·5, and expressed as the amount of enzyme that produces 1 nmol NADPH min1.
Glucose uptake experiments.
A 50 ml volume of the seed culture was used to inoculate a 2·8 l Fernbach flask containing 500 ml of a chemically defined medium (CD medium) with 100 mM D-glucose, as previously reported (Escalante et al., 1999). After 36 h, mycelia (250 mg wet weight) were harvested, washed with 0·85 % NaCl, and resuspended in a vial containing 4·5 ml saline. The suspension was incubated under agitation, and glucose transport was initiated by the addition of 5 µCi (185 kBq) D-[14C]glucose (38·8 MBq mmol1) in 475 µl 10 mM cold glucose, as previously reported (Escalante et al., 1999
). Radioactivity was determined by soaking the filter in vials containing 4 ml of a commercial liquid scintillation counting solution.
Isolation of RNA, and dot blot analyses.
Total RNA of S. peucetius var. caesius was extracted from mycelia of the original strain and its derived DogR mutant, as well as from the DogR mutant transformed with SCO2127. RNA was isolated from cultures grown in YM medium with 100 or 500 mM glucose for 24 h (exponential growth phase) by the method of Chomczynski & Sacchi (1987). Residual DNA was eliminated by incubating the samples with DNase I for 1 h at 37 °C. Total RNA was precipitated by addition of 0·1 vol. 3 M sodium acetate and 1 vol. ice-cold 2-propanol. RNA concentrations were determined spectrophotometrically, and RNA quality was checked by agarose-formamide electrophoresis, according to Kieser et al. (2000)
. RNA was stored at 70 °C for no more than 2 weeks. For dot blot analysis, the RNA concentration was adjusted to 1 µg µl1 with GFP (1 M glyoxal, 10 mM phosphate and 70 % formamide), and then denatured at 55 °C for 15 min. The resulting RNA was kept in an ice-bath until loaded onto Hybond-N+ membranes (Amersham). The amounts of total RNA applied to the membranes were 2, 5, 10 and 20 µg. To detect the glk mRNA, a digoxigenin-UTP-labelled S. peucetius var. caesius glk probe (900 bp) was utilized (DIG High-Prime DNA Labelling and Detection Starter Kit II; Roche). The hybridization assay was carried out overnight at 54 °C. The glk mRNA was developed with an alkaline-phosphataseantidigoxigenin conjugate, and CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate; Roche] as the light emission substrate, and the membrane was exposed to an autoradiography film. Total RNA from Paenibacillus amylolyticus was used as a negative control.
Reproducibility of results.
The results shown are the mean values of at least two independent experiments carried out in triplicate. The observed variations were consistently less than 10 %.
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RESULTS |
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Presence of glkA and SCO2127 homologues in S. peucetius var. caesius
In order to corroborate the presence of the endogenous glkA and SCO2127 genes in S. peucetius var. caesius, we carried out Southern blot experiments using probes obtained from S. coelicolor. As can be seen in Fig. 1, there was good hybridization in S. peucetius var. caesius with both probes, and it was comparable with that found with the S. coelicolor DNA used as the positive control. Additionally, the hybridization profiles found using glkA and SCO2127 were identical to each other regardless of the restriction endonucleases used, showing that these genes are encoded on the same DNA fragment, and thus suggesting that the genomic organization of these genes is similar to that found in S. coelicolor, where they are found adjacent to each other, possibly in the same operon.
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RNA transcription
To gain more information about the stimulatory mechanism of SCO2127, transcription of glk was monitored by performing RNA dot blot experiments. As shown in Fig. 2, when glk mRNA levels from the original strain and a CCR-resistant mutant (DogR) were compared with those of the DogR mutant transformed with SCO2127 (SDR-2), an increase in the glk mRNA levels was observed in the transformed mutant compared with those of the other strains. In contrast, lower glk mRNA levels were obtained in the DogR mutant. In addition, in comparison with cultures grown with 100 mM D-glucose, higher glk mRNA levels were observed when two of the strains (original and DogR mutant) were cultivated with 500 mM D-glucose. On the other hand, these differences were not found in cultures of the DogR mutant transformed with SCO2127. The glucose permease mRNA levels were not determined, since the sequence of this gene was only recently described (Bertram et al., 2004
).
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DISCUSSION |
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Mutants of S. peucetius var. caesius resistant to the inhibition of carbon source utilization by 2-DOG (DogR mutants) exhibit a pleiotropic phenotype, with decreased Glk activity and low glucose incorporation, which seem to cause reversal of glucose repression (Segura et al., 1996; Escalante et al., 1999
). By applying an enrichment procedure to the DogR strain, several mutants were isolated that regained sensitivity to 2-DOG inhibition (DogS). Although sensitive to 2-DOG, these mutants exhibit different glucose uptake/Glk ratios, which render them totally or partially sensitive to CCR (Ramos et al., 2004
). In the present work, we have shown that both DogR and DogS phenotypes can be reverted to that of the original strain by transforming the mutants with plasmid pSG210, which expresses the 620 bp SCO2127 gene of S. coelicolor. In these recombinant strains, glucose uptake and Glk activity values were restored to similar or even higher levels than the original strain, and the recombinants regained sensitivity to CCR. The effect of SCO2127 on Glk activity correlated well with the glk mRNA levels observed in a CCR-resistant mutant transformed with this region. The above-mentioned results were quite unforeseen considering that the SCO2127 region does not seem to code for either a glucose permease (Bertram et al., 2004
) or a glucose kinase (Angell et al., 1994
). Similar results were obtained by transformation with plasmid pIJ513 (Ikeda et al., 1984
), which contains both SCO2127 and glkA. As expected, recombinants with plasmid pSG200 containing a 950 bp fragment with the glkA gene alone reverted to normal Glk activity values, but glucose uptake was unchanged.
Our hybridization experiments suggest that the SCO2127 and glkA genes have a similar genomic organization in both S. coelicolor and S. peucetius var. caesius, or are at least in close proximity within the chromosome. However, there appear to be no data published on the activity and expression of SCO2127 in either of these systems (Angell et al., 1994).
With regard to CCR sensitivity in the mutant strains transformed with either SCO2127 or glkA, anthracycline production showed increased sensitivity to the glucose effect, even at low carbohydrate concentrations (100 mM). This oversensitivity could be explained in terms of a better performance of S. coelicolor SCO2127 and Glk activities in the S. peucetius var. caesius intracellular environment, or by the high-copy-number plasmids harbouring either SCO2127 or glkA. An exception to this observation was obtained with the DogS-2 mutant transformed with glkA. However, this recombinant exhibited about 80 % less glucose incorporation compared to that of the original strain. Therefore, although containing a high Glk activity (threefold higher), its glucose availability is probably very low.
As expected, under glucose-repressive conditions (500 mM), the original strain displayed higher glk mRNA levels than those observed in non-repressive concentrations (100 mM), again supporting the role of Glk activity in CCR sensitivity. These differences were also observed in a mutant resistant to CCR (DogR). In this case, the low glk mRNA levels observed under both repressive and non-repressive conditions correlated well with its CCR-resistant phenotype (Ramos et al., 2004). On the other hand, although higher glk mRNA levels were observed in the DogR strain transformed with SCO2127, no differences were observed in this parameter under repressive and non-repressive conditions, explaining the oversensitivity of this recombinant to the glucose effect.
The question arises of how SCO2127 mediates its stimulating effect on glucose uptake and Glk activity. Our data from the SCO2127-transformed strains do not support the occurrence of recombination between pSG210 and the mutants' chromosomal DNA, thus eliminating the possibility of a highly transcribed SCO2127 promoter as the cause of the stimulatory effect on Glk activity and glucose uptake. In addition, although the glk gene is located next to SCO2127, and in theory may be susceptible to a stimulatory effect from the SCO2127 promoter, the glcP1 (SCO5578) region, which was recently described as encoding a non-PTS glucose permease in S. coelicolor (Bertram et al., 2004), was found to be located not in the vicinity of SCO2127, but on the opposite side of the conserved region of the chromosome. Considering the increase in glk mRNA levels observed in one of the CCR-resistant mutants transformed with SCO2127, we suggest that SCO2127 is involved in stimulating transcription of glk, and probably that of the glucose permease gene as well. In conclusion, our data suggest the participation of an integral regulatory system, which is initiated with an increase in glucose incorporation and metabolism, resulting in an increased synthesis of catabolites, which may be involved in eliciting CCR in this micro-organism. In agreement with this possibility, among several products of glucose metabolism, fructose 1,6-bisphosphate and PEP exert CCR on anthracycline formation in S. peucetius var. caesius (Ramos et al., 2004
), with fructose 1,6-bisphosphate being the most effective. This effect resembles that reported for fructose 1,6-bisphosphate on the phosphorylation of Hpr kinase from Bacillus subtilis as a preliminary step for CCR (Jault et al., 2000
). This would suggest an increase in glycolytic flux as a requisite for the establishment of the phenomenon of CCR in S. peucetius var. caesius.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 August 2004;
revised 23 December 2004;
accepted 7 February 2005.
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