Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-8510, Japan1
Author for correspondence: Kenji Ueda. Tel: +81 466 84 3936. Fax: +81 466 84 3935. e-mail: ueda{at}brs.nihon-u.ac.jp
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ABSTRACT |
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Keywords: Streptomyces griseus, morphological differentiation, carbon source dependence, repressor, craA
Abbreviations: ARP, A-factor receptor protein; MDH, malate dehydrogenase
b The GenBank accession number for the sequence reported in this paper is AB023642.
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INTRODUCTION |
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In this paper, we identify a putative negative regulatory gene for cellular differentiation in S. griseus by an approach opposite to the one that led to the isolation of amfR. We used wild-type S. griseus as a host and cloned genes that, on a high-copy-number plasmid, repressed normal cellular differentiation. This allowed us to clone a DNA fragment that significantly repressed aerial mycelium and spore formation in the presence of glucose and galactose but not in the presence of maltose. Sequence responsible for the repression was identified in the promoter region of a putative negative regulatory gene possibly involved in carbon-source-dependent regulation of cellular differentiation in S. griseus.
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METHODS |
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General recombinant DNA techniques.
Restriction endonucleases and other modifing enzymes were purchased from Takara Shuzo. Thiostrepton was a gift from Asahi Chemical Industry. DNA was manipulated in E. coli as described by Maniatis et al. (1982 ), and in Streptomyces as described by Hopwood et al. (1985
). Nucleotide sequence was determined with an automated DNA sequencer (Licor, model L4000) and a Thermo Sequenase cycle sequencing kit (Amersham).
Shotgun cloning.
Chromosomal DNA isolated from the mycelium of wild-type S. griseus was partially digested with BamHI and ligated to pIJ702 at its BglII site. The ligation mixture was used to transform the wild-type strain, and transformants showing thiostrepton resistance were screened for differentiation on YMP/glucose agar. Colonies showing sporulation-negative phenotypes were cultured in 100 ml YMP/glucose liquid medium. Plasmids were extracted and used to retransform the wild-type to confirm that the phenotype was plasmid-linked.
Subcloning experiments.
The 3·7 kb BamHI fragment originally cloned in pKM284 was at the BglII site of pIJ702. Low-copy-plasmid pKM284L was constructed as follows: the 3·7 kb BamHI region was recovered together with partial sequences from pIJ702 that included the C-terminal half of the thiostrepton resistance gene (tsr) as a 5·9 kb fragment by digesting pKM284 with PstI and EcoRV. This fragment was then inserted between the PstI and EcoRV sites of pIJ922 with the correct junction of the tsr gene. pKM284-1 was constructed by inserting the BamHISphI region between the BglII and SphI sites of pIJ702. pKM284-2, pKM284-2H (see also the next section for promoter assay) and pKM284-2L were constructed by inserting the BamHIFbaI fragment into the BglII site of pIJ702, the BamHI site of pIJ486 and the BamHI site of pIJ922, respectively. pKM284M1 and pKM284M3 were constructed by inserting the BamHIFbaI fragment into the BamHI site of pTMA1 followed by confirmation of their correct orientation by digestion with a combination of BamHI, EcoRI and HindIII. pKM284-3 was constructed by inserting the FbaI fragment containing ORF3 with a partial sequence from pIJ702 into the BglII site of pIJ702. Similarly, the ORF2-containing FbaI fragment was cloned at the BglII site of pIJ702 to generate pKM284-4. To construct pKM284-5, the BalIEcoRI fragment was blunt-ended by treatment with the Klenow fragment, attached to an 8-mer BglII linker at both ends so that it could be recovered as a BglII fragment and cloned at the BglII site of pIJ702. To construct pKM284-6, pKM284 was digested with PmaCI and ligated to an 8-mer PstI linker. The PmaCIKpnI region was recovered as a PstIKpnI fragment and inserted between the PstI and KpnI sites of pIJ702. pKM284-7 was constructed by inserting the PstIKpnI fragment from pKM284-6 between the PstI and KpnI sites of pKM284-4. To construct pKM284-8, the BalIFbaI region was excised as a BglIIFbaI fragment from pKM284-5 and cloned at the BglII site of pIJ702. To construct pKM284-9, the BamHIPmaCI region was excised as a BamHIPstI fragment from pKM284-6 and inserted between the BglII and PstI sites of pIJ702. To construct pKM284-10, the BalIMluI region was excised as a BglIIMluI fragment from pKM284-8 and inserted between the BglII and MluI sites of pIJ702. To construct pKM284-11, the BalIPmaCI fragment was attached to an 8-mer BglII linker, recovered as a BglII fragment and ligated to the BglII site of pIJ702. pKM284-12 was constructed by cloning a trimmed BglII fragment from pKM284-11 at the BglII site of pIJ702. The trimming was done as follows: pKM284-11 was cleaved with SphI and digested with a combination of exonuclease III and mungbean nuclease. After blunt-end formation with the Klenow fragment, an 8-mer BglII linker was attached; the product was digested with BglII and inserted at the BglII site of pIJ702. The trimmed BglII fragment was also cloned at the BamHI site in pUC19 and its nucleotide sequence was determined. To construct pKM2841 and pKM284
3, pKM284 was partially digested with MluI and self-ligated after treatment with the Klenow fragment. The ligation mixture was used to transform the wild-type strain and transformants were screened for colonies harbouring plasmids with the correct frameshifts at the MluI sites.
Promoter assay.
Promoter activities were measured by using a thermostable MDH gene as a reporter, following the method described previously (Vujaklija et al., 1991 ). Plasmids pKM284M1 and pKM284M2, carrying the promoter-less MDH gene preceded by promoters in the direction of ORF1 and ORF3, respectively, were used to transform the S. griseus wild-type strain; MDH activities expressed by those promoters during growth on several media were measured. The strain for assaying the titration effect was constructed by introducing pKM284-2H into a thiostrepton-resistant transformant harbouring pKM284M3, taking advantage of their compatibility. Because of the aphII gene downstream from the inserted promoter, pKM284-2H conferred kanamycin resistance. Transformants showing thiostrepton and kanamycin resistance were screened for their retention of the two kinds of plasmid. Wet weight of mycelium in each culture was measured to assess growth.
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RESULTS |
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One of these plasmids, pKM284, caused significant repression of aerial mycelium and spore formation in wild-type S. griseus grown on YMP/glucose or YMP/galactose agar (Fig. 1a, left). In contrast, it resulted in normal differentiation on YMP/maltose agar, YMP agar without carbon sources (Fig. 1a
) or YMP/mannitol agar (not shown). Neither streptomycin production nor A-factor production was affected on these media. On YMP/glucose agar (Fig. 1b
) pKM284 also repressed aerial mycelium and spore formation by A-factor receptor protein (ARP)-negative mutants of S. griseus HO1 (Onaka et al., 1997
) and KM7 (Miyake et al., 1990
). ARP is a receptor that negatively regulates both cellular differentiation and streptomycin production in S. griseus. Binding of A-factor to ARP results in derepression (Miyake et al., 1990
; Onaka et al., 1997
). The bald phenotype of an A-factor-deficient mutant of S. griseus strain HH1 was not affected by introducing pKM284 (Fig. 1b
, left end). This plasmid also significantly reduced spore formation in S. lividans TK21 on glucose medium (Fig. 1b
, right end).
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Neither the low-copy-number plasmid carrying the original fragment (pKM284L; Fig. 1a, right end) nor other plasmids containing the promoter region on low-copy-number vectors (pKM284-2L, pKM284M1 and pKM284M2; data not shown) caused repression, supporting the idea that the loss of aerial mycelium and spore formation was a multi-copy effect conferred by the high copy number of pIJ702 (40100 copies per genome). Using pIJ486 (40100 copies per genome) to clone the BamHIFbaI region (pKM284-2H) gave the same phenotype as pKM284, indicating that the repressive effect is not specific to pIJ702. Frameshift mutations in ORF1 or ORF3 on pKM284 (pKM284
1 and pKMK284
3) did not abolish the repressive effect of the original plasmid, indicating that the intact forms of neither ORF1 nor ORF3 are essential for repression. We do not have a clear explanation for the slightly leaky repression exhibited by several plasmids, including pKM284-12.
Quantitative analysis of promoter activity and its dependence on carbon source
The above results implying a titration effect of the promoter fragment, along with its dependence on carbon source, prompted us to quantify the promoter activity in vivo. The BamHIFbaI fragment containing the promoter region was cloned in pTMA1, a promoter-probe vector carrying a thermostable MDH gene as a reporter (Vujaklija et al., 1991 ), and the transcriptional activities in the directions of both ORF1 (PORF1) and ORF3 (PORF3) were measured as MDH activities.
As shown in Fig. 4, PORF1 showed relatively low activity throughout the 7 d of growth in YMP liquid media, irrespective of the carbon source. On the other hand, the activity of PORF3 depended markedly on the carbon source: in YMP/glucose, it increased to its highest level after 4 d cultivation and was maintained for a further 3 d. In contrast, in YMP/maltose, it increased during the first 3 d and then dropped sharply. The level during the later cultivation was far lower than that in YMP/glucose. Growth in these cultures was almost identical, with maximum mycelium wet weight at day 5 (Fig. 4a
). With galactose as a sole carbon source the profile was similar to that with glucose, while YMP/mannitol and YMP without additional carbon sources gave similar patterns to that obtained with maltose (not shown). MDH was confirmed to stably reflect promoter activity irrespective of the carbon source in the medium (our unpublished control experiment). Therefore these results suggest that promoter activity in the direction of ORF3 is regulated in a carbon-source-dependent manner.
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DISCUSSION |
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Introducing the promoter region of ORF3 (PORF3) on high-copy-number plasmids caused significant repression of aerial mycelium and spore formation in the presence of glucose or galactose, but not in the presence of maltose or mannitol. Transcriptional activity of PORF3 was enhanced in a carbon-source-dependent manner exactly in parallel with repression of cellular differentiation by pKM284. Elevated transcription from the promoter was maintained until the death phase in the glucose medium, while it dropped sharply during exponential growth in the maltose medium. We assume that this is caused by carbon-source-dependent regulation of PORF3 activity, and that the ORF3 product plays a negative role during initiation of differentiation. ORF3 protein may be produced during vegetative growth to block onset of differentiation, and its carbon-source-dependent repression may initiate differentiation. We propose the name craA to designate the ORF3 gene possibly involved in carbon-source-dependent regulation of aerial mycelium formation. For further characterization of craA as a negative regulator we need to overexpress this gene and disrupt it.
The elevated activities of PORF3 in the presence of pKM284M3 co-existing with pKM284-2H strongly suggested that a transcriptional repressor protein directly binding to the promoter sequence is involved. We detected a DNA-binding protein by gel retardation (data not shown). Binding of this protein to the 249 bp fragment was postulated to block the onset of differentiation during vegetative growth; its carbon-source-dependent transcriptional repression caused by the DNA-binding protein initiates aerial mycelium formation. The concentration of glucose in the medium might also be a key factor affecting the onset of differentiation through repression of craA transcription.
pKM284 repressed aerial mycelium and spore formation of ARP-negative mutants. This indicates that the regulatory point of craA is not directly related to ARP function, but is probably located in a downstream regulatory pathway specific to morphological differentiation. We could not detect a difference in PORF3 activity between an A-factor-deficient mutant and a wild-type strain of S. griseus (data not shown). This suggests that craA-mediated regulation acts independently of the A-factor cascade and integration of the signals from both systems may initiate differentiation. Evidence of significant repression by pKM284 in S. lividans (Fig. 1b) as well as in S. griseus suggests that craA-mediated regulation may be generally distributed among streptomycetes.
Carbon-source-dependent regulation of the initiation of differentiation has been suggested through the phenotypic features of bld mutants of S. coelicolor A3(2). Several classes of bld mutants, including bldA and bldD (Chater, 1989 ), as well as cya, an adenylate cyclase mutant (Süsstrunk et al., 1998
), are known to show restored aerial mycelium on mannitol medium. In wild-type S. griseus, we observed that the efficiency of aerial mycelium and spore formation depends on carbon sources in the media. S. griseus shows abundant sporulation on maltose or mannitol media, and relatively poor and delayed sporulation on glucose media. Availability of carbon sources may be one of the factors that determine the timing of cellular differentiation in streptomycetes, and the putative regulatory system revealed in this study could be directly involved in one such carbon-source-dependent control mechanism.
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ACKNOWLEDGEMENTS |
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Received 3 March 1999;
revised 26 May 1999;
accepted 15 June 1999.
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