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Cloning of the rplA gene encoding ribosomal protein L1 from Streptomyces aureofaciens and its transcriptional analysis in the course of differentiation
FEMS Microbiology Letters 178 (1999) 129^134
Cloning of the rplA gene encoding ribosomal protein L1 from Streptomyces aureofaciens and its transcriptional analysis in the course of di¡erentiation Kormanec Ja¨n *, Nova¨kova¨ Rena¨ta, Kl'uca¨r L'ubos, Home¨rova¨ Dagmar, Síevc¨|kova¨ Beatrica, Sprusansky¨ Ondrej Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska¨ cesta 21, 842 51 Bratislava, Slovak Republic Received 10 May 1999; received in revised form 5 July 1999; accepted 9 July 1999
Abstract Using the method for the identification of promoters recognized by the sporulation-specific c factor RpoZ, we identified a positive 882-bp Sau3AI DNA fragment in Streptomyces aureofaciens. Sequence analysis revealed a complete open reading frame encoding a protein of 242 amino acids with a Mr of 25974. The deduced protein product showed high sequence similarity to all ribosomal proteins L1, with maximum similarity to several Streptomyces spp. L1 (97% identity). The 5P-truncated rplK gene encoding ribosomal protein L11 was located upstream of the rplA gene. However, the putative rpoZ-dependent promoter, Pren25 , was located divergently in the coding region of the rplA gene and was inactive in S. aureofaciens. To investigate expression of the rplA gene, Northern blot hybridization was performed using RNA prepared from S. aureofaciens in various developmental stages. These studies suggested that the rplA gene is expressed in all stages of differentiation as a 1400nucleotides transcript. The amount of the transcript decreased during differentiation. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ribosomal protein; Di¡erentiation ; Northern blot; Transcription; Streptomyces aureofaciens
1. Introduction Streptomycetes are Gram-positive mycelial soil bacteria that undergo a complex process of morphological di¡erentiation, which involves the formation of spore-bearing aerial hyphae on mycelial colonies [1]. During the process of morphological di¡erentiation, di¡erences in the pattern of ribosomal proteins
* Corresponding author. Tel.: +421 (7) 5941 2432; Fax: +421 (7) 54772316; E-mail: [email protected]
have been described for Streptomyces [2]. The di¡erences were also described for a liquid-grown Streptomyces coelicolor strain, where the synthesis of all ribosomal proteins decreased at the approach of the transition phase, that is characteristic by the decrease of the growth rate, and preceded the stationary phase [3]. Several genes encoding Streptomyces ribosomal proteins have been cloned and sequenced. They comprise the rplJL operon encoding ribosomal protein L7/L12 in Streptomyces antibioticus [4] and in S. coelicolor [3], the rplM, rpsI operon encoding L13, S9 in S. coelicolor [5], the rplKA and rplJL
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 5 0 - X
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operons encoding L11, L1 and L7/12, L10, respectively, in Streptomyces griseus [6]. However, the transcriptional analysis has only been performed in the rplM, rpsI operon in S. coelicolor [5] and with rplKA and rplJL operons in S. griseus [6]. In both cases, it was performed only during the growth in liquid medium. There are no transcriptional studies of ribosomal protein genes in the course of di¡erentiation of streptomycetes. Previously, we established a method for the identi¢cation of Streptomyces promoters recognized by a particular Streptomyces c factor of RNA polymerase [7]. The method is based on two Escherichia coli compatible plasmids. A particular Streptomyces c factor heterologously expressed from one plasmid (expression plasmid pAC5mut2) can interact with the E. coli RNA polymerase core enzyme and the resulting heterologous holoenzyme recognizes a promoter present in a library of Streptomyces chromosomal fragments cloned in the second compatible plasmid (promoter probe plasmid pSB40), upstream of a promoterless lacZK reporter gene. After induction with IPTG and selection on X-Gal plates, positive promoters are identi¢ed as blue colonies only in the presence of IPTG-induced expression of a particular c factor gene [7]. Using the method, we identi¢ed a new promoter, Pren40 , recognized by sporulation-speci¢c c factor RpoZ [7]. While searching for all possible Streptomyces aureofaciens promoters recognized by RpoZ, we identi¢ed six independent clones being positive only in the background of rpoZ-encoded c factor. In one case, sequence analysis of a positive fragment identi¢ed an open reading frame (ORF), located divergently to the proposed RpoZ-dependent promoter, encoding a deduced protein with high sequence similarity (97% identity) to ribosomal proteins L1. The present study describes, in addition to sequence analysis of the rplA gene, the transcriptional analysis of the gene in the course of di¡erentiation.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions S. aureofaciens CCM3239 (ATCC 10762) was from the Czechoslovak Collection of Microorgan-
isms (Brno, Czech Republic). E. coli XL1Blue (Stratagene) was used as host and plasmid pBluescript II SK+ (Stratagene) was used for E. coli cloning experiments. E. coli promoter probe plasmid pSB40 [8] was kindly provided by Dr M.K. Winson, University of Nottingham, UK. For RNA isolation from surface culture, 108 colony forming units of S. aureofaciens wild-type strain were spread on sterile cellophane membranes placed on Bennet medium [9] and grown to the appropriate phase of development. Conditions for E. coli growth and transformation were as described [10]. 2.2. DNA manipulations DNA manipulations in E. coli were done as described in [10] and those in Streptomyces were as described in [11]. DNA fragments were isolated from an agarose gel by the GeneClean technique (BIO 101, LaJolla, CA, USA). Nucleotide sequencing was performed by the chemical method [12]. Compressions caused by a high percentage of G-C pairs were removed by running gels equilibrated at 75³C. 2.3. RNA isolation and Northern blot hybridization Total RNA was prepared from surface cultures of S. aureofaciens as previously described [13] and dissolved in deionized formamide. 10 Wg of RNA was electrophoresed in a 1.2% (w/v) agarose gel containing 2.2 M formaldehyde and RNA was transferred on a Hybond N membrane (Amersham) as described by [10]. The membrane was hybridized with a DNA probe in EasyHyb hybridization solution (Boehringer) at 45³C. The DNA probe was labelled with digoxigenin (DIG) by the random-primed DIGDNA labelling kit (Boehringer) and used in a concentration of 20 Wg ml31 hybridization solution. After hybridization, the blot was washed according to the standard DIG protocol and a signal was detected by the DIG chemiluminescent detection kit using CSPD (Boehringer). The membrane was immediately exposed to an X-ray ¢lm. 2.4. S1 nuclease mapping High-resolution S1 nuclease mapping was per-
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Fig. 1. A: Restriction map of the 882-bp positive Sau3AI fragment of plasmid pREN25 containing the rplA gene (black box) and the 3Pend of the rplK gene (hatched box) of S. aureofaciens, cloned in the BamHI site of promoter probe plasmid pSB40. The open circle represents the position of a putative RpoZ-dependent promoter Pren25 , active only in the E. coli two-plasmid system. The stippled box below the map represents the probe used for Northern hybridization analysis. The thin lines below the map represent DNA fragments (5P-labelled at the end marked with an asterisk) that were used as probes in S1 nuclease mapping. Thick lines under the map represent deletions of the fragment with the resulting phenotype indicating rpoZ-dependent promoter activity. Relevant restriction sites are indicated. The nucleotide sequence was deposited at GenBank under the accession number AF120458. B: Comparison of the putative Pren25 promoter with the previously identi¢ed S. aureofaciens rpoZ-dependent promoters Pren40 and PwhiH from S. aureofaciens [7,16]. Identical nucleotides are highlighted in black. Nsubscript indicates the number of intervening nucleotides. The designation (+1) indicates the transcription start point.
formed as described previously [14]. Samples (40 Wg) of RNA were hybridized to approximately 0.1 pmol suitable DNA probe labelled at one 5P-end with [Q32 P]ATP (approximately 106 cpm pmol31 probe). The P2hrdB promoter has been described by [14]. The protected DNA fragments were analyzed on DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments [12].
3. Results and discussion 3.1. Cloning of the S. aureofaciens chromosomal fragment containing the rplA gene Previously, we established a method for the identi¢cation of Streptomyces promoters recognized by a particular Streptomyces c factor of RNA polymerase [7]. We have used the method for screening of all
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Fig. 2. Northern blot hybridization analysis of the rplA transcription in the course of di¡erentiation with the ApaI-XhoI internal DNA probe of rplA (Fig. 1A). Lane 1, RNA from S. aureofaciens grown on solid Bennet medium for 13 h, substrate mycelium. Lane 2, 19 h, beginning of aerial mycelium formation. Lane 3, 36 h, aerial mycelium approximately in the time of septation. Lane 4, 60 h, spores. Positions of the internal rRNAs are denoted by lines. The size of the transcript was calculated using the rRNAs as internal standards assuming that their sizes are as in Streptomyces ambofaciens : 23S rRNA = 3120 nucleotides, 16S rRNA = 1528 nucleotides [19].
possible S. aureofaciens promoters recognized by sporulation c factor RpoZ [15]. The S. aureofaciens CCM 3239 genomic library of 0.7^1.2-kb partial Sau3AI chromosomal fragments in the BamHI site of promoter probe vector pSB40 [8] was transformed into E. coli containing the compatible plasmid pACrpoZ, having the rpoZ gene under the control of a strong IPTG-inducible trc promoter [7]. After screening of about 60 000 colonies, we identi¢ed six independent clones being positive only in the background of rpoZ-encoded c factor (E. coli containing pACrpoZ). In addition to the previously identi¢ed promoters, Pren40 directing the sporulation gene ren40 [7] and PwhiH directing the gene for the sporulation transcription factor whiH [16], in one case (plasmid pREN25 carrying an 882-bp Sau3AI DNA fragment, Fig. 1A), sequence analysis revealed a complete ORF having codon usage typical for Streptomyces genes with a marked preference for G or C (90.4%) in the third nucleotide of the triplet [17]. This codon usage with the preference for U in the third position for several codons is typical for highly expressed genes in Streptomyces [17]. Similar codon usage was also
found in other streptomycetes ribosomal protein genes [4,6]. The ORF encodes a deduced protein of 242 amino acids (Mr 25 974). Comparison of the deduced protein with data bases revealed end to end extensive homology to all ribosomal proteins L1 encoded by the rplA gene, with maximum similarity to several Streptomyces spp. L1. All Streptomyces L1 proteins shared 88% identical amino acids. The pairwise amino acids identity between the particular genes varies from 88 to 97.5%. Sequence analysis of the 882-bp cloned fragment revealed that the 5P-truncated rplK gene encoding ribosomal protein L11 was located upstream of the rplA gene. This organization is common to all identi¢ed ribosomal proteins L1 and L11 in bacteria, where these two genes are organized in operon rplKA [6]. All 15 Cterminal amino acids of S. aureofaciens ribosomal protein L11 were identical to their counterparts in other Streptomyces spp. 3.2. Analysis of a putative RpoZ-dependent promoter Pren25 Based on the direction of expression of a putative rpoZ-dependent promoter to the lacZK reporter gene of the plasmid pREN25, the orientation of the rplA gene is divergent to the putative rpoZ-dependent promoter. The deletion analysis of the fragment (Fig. 1A) and sequence analysis revealed the sequence, TCAT-N16 -GCCGATGA in the coding region of rplA (Fig. 1B), having high similarity to the consensus sequence of promoters recognized by £agellar and chemotaxis c factors cD of Bacillus subtilis and cF of Salmonella typhimurium [18] and also to recently identi¢ed promoters Pren40 and PwhiH recognized by S. aureofaciens rpoZ-encoded c factor [7,16]. We named the putative promoter Pren25 . Comparison of all three S. aureofaciens RpoZ-dependent promoters is shown in Fig. 1B. In order to examine the expression of the putative Pren25 promoter in S. aureofaciens, high-resolution S1 nuclease mapping was performed using a 5P-labelled probe at the single NotI site in the rplA coding region (Fig. 1A) and RNA isolated from a surface-grown culture of S. aureofaciens during di¡erentiation on solid Bennet medium. In repeated experiments, we did not detect any protected fragment with the probe, whereas a control probe for the P2hrdB promoter [14] gave con-
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sistent expression levels using the same RNA samples (data not shown). The results of the transcriptional analysis have revealed that the Pren25 promoter is not expressed in any stage of di¡erentiation. Pren25 has promoter activity only in E. coli in the presence of IPTG-induced rpoZ expression (E. coli containing pAC-rpoZ). In spite of constituting an RpoZ-cognate sequence, the Pren25 is not active in S. aureofaciens. Taking together, these results and the Pren25 divergent location in the rplA coding region, it seems very unlikely that it constitutes a functional promoter in S. aureofaciens. Thus, caution should be taken when using the system for identi¢cation of promoters dependent upon a particular c factor [7]. The identi¢ed promoters must always be identi¢ed in an appropriate host strain by S1 nuclease mapping or in vivo promoter probing. 3.3. Expression of the S. aureofaciens rplA gene in the course of di¡erentiation In spite of the cloning of several genes encoding ribosomal proteins in Streptomyces, there is no information about the expression of these genes at the di¡erent developmental stages. To examine the expression of rplA in the course of di¡erentiation, Northern blot hybridization was performed with RNA isolated from surface-grown S. aureofaciens and an internal DNA fragment of the rplA gene as a probe (590-bp ApaI-XhoI DNA fragment from plasmid pREN25, Fig. 1A). The results revealed a unique signal of about 1400 nucleotides (Fig. 2), detected with the RNA isolated from all stages, having a higher intensity with the RNA from vegetative substrate mycelium. The length of the transcript (1400 nucleotides) was much higher than the estimated size of the rplA gene (730 bp). A signal of similar length was identi¢ed in parallel Northern blot hybridization using a DNA probe covering the whole 882-bp Sau3AI fragment from pREN25 (Fig. 1A) (data not shown). This assumes that the S. aureofaciens rplA gene is expressed as a polycistronic mRNA, likely as bicistronic, with the upstream rplK gene. To prove that the transcript is not starting in the rplA-rplK intergenic region, S1 nuclease mapping was performed with the identical RNA isolated from surface-grown S. aureofaciens and a 5P-labelled DNA probe at the single XhoI site in the rplA coding
133
region (Fig. 1A). No transcription start point was identi¢ed with any RNA in the intergenic region. However, the whole DNA probe was RNA-protected, with a decreased intensity, similar to the results of Northern blot hybridization (data not shown). This indicates that the identi¢ed bicistronic transcript is starting upstream of rplK, forming the rplKA operon. These results are similar to S. griseus, where a transcript of similar length (approximately 1400 nucleotides) covering the rplKA operon was identi¢ed [6]. However, the expression of the rplKA operon in S. griseus is di¡erent. There were no signi¢cant di¡erences in the amount of rplKA transcript in the various growth phases during cultivation of S. griseus in liquid minimal medium [6]. On the contrary, the expression of all ribosomal proteins, as measured by protein pulse-labelling experiments in S. coelicolor, decreased along the cell cycle in liquid-grown culture and the synthesis of at least two ribosomal proteins (L7/L12 and L10) stopped after 20^24 h of growth [3]. A similar decrease of expression along the cell cycle was found for the operon containing rplM and rpsI genes encoding ribosomal proteins ScoL13 and ScoS9 in S. coelicolor [5]. However, all these studies were performed with the liquid-grown cells and there are no data about the expression of ribosomal protein genes in Streptomyces at the di¡erent developmental stages during the morphological di¡erentiation in solid-grown cultures. The S. aureofaciens rplA gene, as a bicistronic transcript with the upstream rplK gene, is transcribed during all stages of di¡erentiation and the transcription decreased in the course of di¡erentiation compared to vegetative substrate mycelium. Our results are also consistent with the analysis of the ribosomal protein pattern during the morphological di¡erentiation of S. antibioticus, where a decrease of several ribosomal proteins was observed in aerial mycelium, including a strong decrease in the L7/L12 content [2,4].
Acknowledgements We would like to thank Mrs. Rena¨ta Knirschova¨ for excellent technical assistance. This work was supported by a Grant 2/4007/98 from the Slovak Academy of Sciences.
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References [1] Chater, K.F. (1993) Genetics of di¡erentiation in Streptomyces. Annu. Rev. Microbiol. 47, 685^713. [2] Quiros, L.M., Parra, F. and Salas, J.A. (1992) Changes in ribosomal proteins during colony development in Streptomyces. Can. J. Microbiol. 38, 1260^1269. [3] Blanco, G., Rodicio, M.R., Puglia, A.M., Mendez, C., Thompson, C.J. and Salas, J.A. (1994) Synthesis of ribosomal proteins during growth of Streptomyces coelicolor. Mol. Microbiol. 12, 375^385. [4] Parra, F., Blanco, G., Alonso, J.M.M., Balbin, M., Mendez, C. and Salas, J.A. (1992) Cloning and sequence of a gene encoding the L7/L12 ribosomal protein equivalent of Streptomyces antibioticus. Gene 118, 127^129. [5] Sanchez, C., Blanco, G., Mendez, C. and Salas, J.A. (1997) Cloning, sequencing and transcriptional analysis of a Streptomyces coelicolor operon containing the rplM and rpsI genes encoding ribosomal proteins ScoL13 and ScoS9. Mol. Gen. Genet. 257, 91^96. [6] Kuster, C., Piepersberg, W. and Distler, J. (1998) Cloning and transcriptional analysis of the rplKA- orf31- rplJL gene cluster of Streptomyces griseus. Mol. Gen. Genet. 257, 219^229. [7] Nova¨kova¨, R., Síevc¨|kova¨, B. and Kormanec, J. (1998) A method for the identi¢cation of promoters recognized by RNA polymerase containing a particular sigma factor: Cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ. Gene 208, 43^50. [8] Park, S.F., Stirling, D.A., Hulton, C.S.J., Booth, I.R., Higgins, C.F. and Stewart, G.S.A.B. (1989) A novel, non-invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12. Mol. Microbiol. 3, 1011^ 1023. [9] Horinouchi, S., Hara, O. and Beppu, T. (1983) Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor
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A3 and Streptomyces lividans. J. Bacteriol. 155 (2), 1238^ 1248. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K. (1987) Current Protocols in Molecular Biology. Wiley, New York. Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.L., Smith, C.P., Ward, J.M. and Schrempf, H. (1985) Genetic Manipulation of Streptomyces. A laboratory Manual. The John Innes Foundation, Norwich. Maxam, A.M. and Gilbert, W. (1980) Sequencing end-labelled DNA with base speci¢c chemical cleavages. Methods Enzymol. 65, 449^560. Kormanec, J. and Farkasovsky¨, M. (1994) Isolation of total RNA from yeast and bacteria and detection of rRNA in Northern blots. BioTechniques 17, 838^842. Kormanec, J. and Farkasovsky¨, M. (1993) Di¡erential expression of principal c factor homologues of Streptomyces aureofaciens correlates with the developmental stage. Nucleic Acids Res. 21, 3647^3652. í ezuchova¨, B. (1994) The Kormanec, J., Potu¨ckova¨, L. and R Streptomyces aureofaciens homologue of the whiG encoding a putative sigma factor essential for sporulation. Gene 143, 101^ 103. Kormanec, J., Nova¨kova¨, R., Homerova¨, D. and Síevc¨|kova¨, B. (1999) The Streptomyces aureofaciens homologue of the sporulation gene whiH is dependent on rpoZ-encoded c factor. Biochim. Biophys. Acta 1444, 80^84. Wright, F. and Bibb, M. (1992) Codon usage in the G+C rich Streptomyces genome. Gene 113, 55^65. Helmann, J.D. (1991) Alternative sigma factors and the regulation of £agellar gene expression. Mol. Microbiol. 5, 2875^ 2882. Pernodet, J.L., Boccard, F., Alegre, M.T., Gagnat, J. and Guerineau, M. (1989) Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens. Gene 79, 33^46.
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Cloning of the rplA gene encoding ribosomal protein L1 from Streptomyces aureofaciens and its transcriptional analysis in the course of di¡erentiation Kormanec Ja¨n *, Nova¨kova¨ Rena¨ta, Kl'uca¨r L'ubos, Home¨rova¨ Dagmar, Síevc¨|kova¨ Beatrica, Sprusansky¨ Ondrej Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska¨ cesta 21, 842 51 Bratislava, Slovak Republic Received 10 May 1999; received in revised form 5 July 1999; accepted 9 July 1999
Abstract Using the method for the identification of promoters recognized by the sporulation-specific c factor RpoZ, we identified a positive 882-bp Sau3AI DNA fragment in Streptomyces aureofaciens. Sequence analysis revealed a complete open reading frame encoding a protein of 242 amino acids with a Mr of 25974. The deduced protein product showed high sequence similarity to all ribosomal proteins L1, with maximum similarity to several Streptomyces spp. L1 (97% identity). The 5P-truncated rplK gene encoding ribosomal protein L11 was located upstream of the rplA gene. However, the putative rpoZ-dependent promoter, Pren25 , was located divergently in the coding region of the rplA gene and was inactive in S. aureofaciens. To investigate expression of the rplA gene, Northern blot hybridization was performed using RNA prepared from S. aureofaciens in various developmental stages. These studies suggested that the rplA gene is expressed in all stages of differentiation as a 1400nucleotides transcript. The amount of the transcript decreased during differentiation. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ribosomal protein; Di¡erentiation ; Northern blot; Transcription; Streptomyces aureofaciens
1. Introduction Streptomycetes are Gram-positive mycelial soil bacteria that undergo a complex process of morphological di¡erentiation, which involves the formation of spore-bearing aerial hyphae on mycelial colonies [1]. During the process of morphological di¡erentiation, di¡erences in the pattern of ribosomal proteins
* Corresponding author. Tel.: +421 (7) 5941 2432; Fax: +421 (7) 54772316; E-mail: [email protected]
have been described for Streptomyces [2]. The di¡erences were also described for a liquid-grown Streptomyces coelicolor strain, where the synthesis of all ribosomal proteins decreased at the approach of the transition phase, that is characteristic by the decrease of the growth rate, and preceded the stationary phase [3]. Several genes encoding Streptomyces ribosomal proteins have been cloned and sequenced. They comprise the rplJL operon encoding ribosomal protein L7/L12 in Streptomyces antibioticus [4] and in S. coelicolor [3], the rplM, rpsI operon encoding L13, S9 in S. coelicolor [5], the rplKA and rplJL
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 5 0 - X
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operons encoding L11, L1 and L7/12, L10, respectively, in Streptomyces griseus [6]. However, the transcriptional analysis has only been performed in the rplM, rpsI operon in S. coelicolor [5] and with rplKA and rplJL operons in S. griseus [6]. In both cases, it was performed only during the growth in liquid medium. There are no transcriptional studies of ribosomal protein genes in the course of di¡erentiation of streptomycetes. Previously, we established a method for the identi¢cation of Streptomyces promoters recognized by a particular Streptomyces c factor of RNA polymerase [7]. The method is based on two Escherichia coli compatible plasmids. A particular Streptomyces c factor heterologously expressed from one plasmid (expression plasmid pAC5mut2) can interact with the E. coli RNA polymerase core enzyme and the resulting heterologous holoenzyme recognizes a promoter present in a library of Streptomyces chromosomal fragments cloned in the second compatible plasmid (promoter probe plasmid pSB40), upstream of a promoterless lacZK reporter gene. After induction with IPTG and selection on X-Gal plates, positive promoters are identi¢ed as blue colonies only in the presence of IPTG-induced expression of a particular c factor gene [7]. Using the method, we identi¢ed a new promoter, Pren40 , recognized by sporulation-speci¢c c factor RpoZ [7]. While searching for all possible Streptomyces aureofaciens promoters recognized by RpoZ, we identi¢ed six independent clones being positive only in the background of rpoZ-encoded c factor. In one case, sequence analysis of a positive fragment identi¢ed an open reading frame (ORF), located divergently to the proposed RpoZ-dependent promoter, encoding a deduced protein with high sequence similarity (97% identity) to ribosomal proteins L1. The present study describes, in addition to sequence analysis of the rplA gene, the transcriptional analysis of the gene in the course of di¡erentiation.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions S. aureofaciens CCM3239 (ATCC 10762) was from the Czechoslovak Collection of Microorgan-
isms (Brno, Czech Republic). E. coli XL1Blue (Stratagene) was used as host and plasmid pBluescript II SK+ (Stratagene) was used for E. coli cloning experiments. E. coli promoter probe plasmid pSB40 [8] was kindly provided by Dr M.K. Winson, University of Nottingham, UK. For RNA isolation from surface culture, 108 colony forming units of S. aureofaciens wild-type strain were spread on sterile cellophane membranes placed on Bennet medium [9] and grown to the appropriate phase of development. Conditions for E. coli growth and transformation were as described [10]. 2.2. DNA manipulations DNA manipulations in E. coli were done as described in [10] and those in Streptomyces were as described in [11]. DNA fragments were isolated from an agarose gel by the GeneClean technique (BIO 101, LaJolla, CA, USA). Nucleotide sequencing was performed by the chemical method [12]. Compressions caused by a high percentage of G-C pairs were removed by running gels equilibrated at 75³C. 2.3. RNA isolation and Northern blot hybridization Total RNA was prepared from surface cultures of S. aureofaciens as previously described [13] and dissolved in deionized formamide. 10 Wg of RNA was electrophoresed in a 1.2% (w/v) agarose gel containing 2.2 M formaldehyde and RNA was transferred on a Hybond N membrane (Amersham) as described by [10]. The membrane was hybridized with a DNA probe in EasyHyb hybridization solution (Boehringer) at 45³C. The DNA probe was labelled with digoxigenin (DIG) by the random-primed DIGDNA labelling kit (Boehringer) and used in a concentration of 20 Wg ml31 hybridization solution. After hybridization, the blot was washed according to the standard DIG protocol and a signal was detected by the DIG chemiluminescent detection kit using CSPD (Boehringer). The membrane was immediately exposed to an X-ray ¢lm. 2.4. S1 nuclease mapping High-resolution S1 nuclease mapping was per-
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Fig. 1. A: Restriction map of the 882-bp positive Sau3AI fragment of plasmid pREN25 containing the rplA gene (black box) and the 3Pend of the rplK gene (hatched box) of S. aureofaciens, cloned in the BamHI site of promoter probe plasmid pSB40. The open circle represents the position of a putative RpoZ-dependent promoter Pren25 , active only in the E. coli two-plasmid system. The stippled box below the map represents the probe used for Northern hybridization analysis. The thin lines below the map represent DNA fragments (5P-labelled at the end marked with an asterisk) that were used as probes in S1 nuclease mapping. Thick lines under the map represent deletions of the fragment with the resulting phenotype indicating rpoZ-dependent promoter activity. Relevant restriction sites are indicated. The nucleotide sequence was deposited at GenBank under the accession number AF120458. B: Comparison of the putative Pren25 promoter with the previously identi¢ed S. aureofaciens rpoZ-dependent promoters Pren40 and PwhiH from S. aureofaciens [7,16]. Identical nucleotides are highlighted in black. Nsubscript indicates the number of intervening nucleotides. The designation (+1) indicates the transcription start point.
formed as described previously [14]. Samples (40 Wg) of RNA were hybridized to approximately 0.1 pmol suitable DNA probe labelled at one 5P-end with [Q32 P]ATP (approximately 106 cpm pmol31 probe). The P2hrdB promoter has been described by [14]. The protected DNA fragments were analyzed on DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments [12].
3. Results and discussion 3.1. Cloning of the S. aureofaciens chromosomal fragment containing the rplA gene Previously, we established a method for the identi¢cation of Streptomyces promoters recognized by a particular Streptomyces c factor of RNA polymerase [7]. We have used the method for screening of all
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Fig. 2. Northern blot hybridization analysis of the rplA transcription in the course of di¡erentiation with the ApaI-XhoI internal DNA probe of rplA (Fig. 1A). Lane 1, RNA from S. aureofaciens grown on solid Bennet medium for 13 h, substrate mycelium. Lane 2, 19 h, beginning of aerial mycelium formation. Lane 3, 36 h, aerial mycelium approximately in the time of septation. Lane 4, 60 h, spores. Positions of the internal rRNAs are denoted by lines. The size of the transcript was calculated using the rRNAs as internal standards assuming that their sizes are as in Streptomyces ambofaciens : 23S rRNA = 3120 nucleotides, 16S rRNA = 1528 nucleotides [19].
possible S. aureofaciens promoters recognized by sporulation c factor RpoZ [15]. The S. aureofaciens CCM 3239 genomic library of 0.7^1.2-kb partial Sau3AI chromosomal fragments in the BamHI site of promoter probe vector pSB40 [8] was transformed into E. coli containing the compatible plasmid pACrpoZ, having the rpoZ gene under the control of a strong IPTG-inducible trc promoter [7]. After screening of about 60 000 colonies, we identi¢ed six independent clones being positive only in the background of rpoZ-encoded c factor (E. coli containing pACrpoZ). In addition to the previously identi¢ed promoters, Pren40 directing the sporulation gene ren40 [7] and PwhiH directing the gene for the sporulation transcription factor whiH [16], in one case (plasmid pREN25 carrying an 882-bp Sau3AI DNA fragment, Fig. 1A), sequence analysis revealed a complete ORF having codon usage typical for Streptomyces genes with a marked preference for G or C (90.4%) in the third nucleotide of the triplet [17]. This codon usage with the preference for U in the third position for several codons is typical for highly expressed genes in Streptomyces [17]. Similar codon usage was also
found in other streptomycetes ribosomal protein genes [4,6]. The ORF encodes a deduced protein of 242 amino acids (Mr 25 974). Comparison of the deduced protein with data bases revealed end to end extensive homology to all ribosomal proteins L1 encoded by the rplA gene, with maximum similarity to several Streptomyces spp. L1. All Streptomyces L1 proteins shared 88% identical amino acids. The pairwise amino acids identity between the particular genes varies from 88 to 97.5%. Sequence analysis of the 882-bp cloned fragment revealed that the 5P-truncated rplK gene encoding ribosomal protein L11 was located upstream of the rplA gene. This organization is common to all identi¢ed ribosomal proteins L1 and L11 in bacteria, where these two genes are organized in operon rplKA [6]. All 15 Cterminal amino acids of S. aureofaciens ribosomal protein L11 were identical to their counterparts in other Streptomyces spp. 3.2. Analysis of a putative RpoZ-dependent promoter Pren25 Based on the direction of expression of a putative rpoZ-dependent promoter to the lacZK reporter gene of the plasmid pREN25, the orientation of the rplA gene is divergent to the putative rpoZ-dependent promoter. The deletion analysis of the fragment (Fig. 1A) and sequence analysis revealed the sequence, TCAT-N16 -GCCGATGA in the coding region of rplA (Fig. 1B), having high similarity to the consensus sequence of promoters recognized by £agellar and chemotaxis c factors cD of Bacillus subtilis and cF of Salmonella typhimurium [18] and also to recently identi¢ed promoters Pren40 and PwhiH recognized by S. aureofaciens rpoZ-encoded c factor [7,16]. We named the putative promoter Pren25 . Comparison of all three S. aureofaciens RpoZ-dependent promoters is shown in Fig. 1B. In order to examine the expression of the putative Pren25 promoter in S. aureofaciens, high-resolution S1 nuclease mapping was performed using a 5P-labelled probe at the single NotI site in the rplA coding region (Fig. 1A) and RNA isolated from a surface-grown culture of S. aureofaciens during di¡erentiation on solid Bennet medium. In repeated experiments, we did not detect any protected fragment with the probe, whereas a control probe for the P2hrdB promoter [14] gave con-
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sistent expression levels using the same RNA samples (data not shown). The results of the transcriptional analysis have revealed that the Pren25 promoter is not expressed in any stage of di¡erentiation. Pren25 has promoter activity only in E. coli in the presence of IPTG-induced rpoZ expression (E. coli containing pAC-rpoZ). In spite of constituting an RpoZ-cognate sequence, the Pren25 is not active in S. aureofaciens. Taking together, these results and the Pren25 divergent location in the rplA coding region, it seems very unlikely that it constitutes a functional promoter in S. aureofaciens. Thus, caution should be taken when using the system for identi¢cation of promoters dependent upon a particular c factor [7]. The identi¢ed promoters must always be identi¢ed in an appropriate host strain by S1 nuclease mapping or in vivo promoter probing. 3.3. Expression of the S. aureofaciens rplA gene in the course of di¡erentiation In spite of the cloning of several genes encoding ribosomal proteins in Streptomyces, there is no information about the expression of these genes at the di¡erent developmental stages. To examine the expression of rplA in the course of di¡erentiation, Northern blot hybridization was performed with RNA isolated from surface-grown S. aureofaciens and an internal DNA fragment of the rplA gene as a probe (590-bp ApaI-XhoI DNA fragment from plasmid pREN25, Fig. 1A). The results revealed a unique signal of about 1400 nucleotides (Fig. 2), detected with the RNA isolated from all stages, having a higher intensity with the RNA from vegetative substrate mycelium. The length of the transcript (1400 nucleotides) was much higher than the estimated size of the rplA gene (730 bp). A signal of similar length was identi¢ed in parallel Northern blot hybridization using a DNA probe covering the whole 882-bp Sau3AI fragment from pREN25 (Fig. 1A) (data not shown). This assumes that the S. aureofaciens rplA gene is expressed as a polycistronic mRNA, likely as bicistronic, with the upstream rplK gene. To prove that the transcript is not starting in the rplA-rplK intergenic region, S1 nuclease mapping was performed with the identical RNA isolated from surface-grown S. aureofaciens and a 5P-labelled DNA probe at the single XhoI site in the rplA coding
133
region (Fig. 1A). No transcription start point was identi¢ed with any RNA in the intergenic region. However, the whole DNA probe was RNA-protected, with a decreased intensity, similar to the results of Northern blot hybridization (data not shown). This indicates that the identi¢ed bicistronic transcript is starting upstream of rplK, forming the rplKA operon. These results are similar to S. griseus, where a transcript of similar length (approximately 1400 nucleotides) covering the rplKA operon was identi¢ed [6]. However, the expression of the rplKA operon in S. griseus is di¡erent. There were no signi¢cant di¡erences in the amount of rplKA transcript in the various growth phases during cultivation of S. griseus in liquid minimal medium [6]. On the contrary, the expression of all ribosomal proteins, as measured by protein pulse-labelling experiments in S. coelicolor, decreased along the cell cycle in liquid-grown culture and the synthesis of at least two ribosomal proteins (L7/L12 and L10) stopped after 20^24 h of growth [3]. A similar decrease of expression along the cell cycle was found for the operon containing rplM and rpsI genes encoding ribosomal proteins ScoL13 and ScoS9 in S. coelicolor [5]. However, all these studies were performed with the liquid-grown cells and there are no data about the expression of ribosomal protein genes in Streptomyces at the di¡erent developmental stages during the morphological di¡erentiation in solid-grown cultures. The S. aureofaciens rplA gene, as a bicistronic transcript with the upstream rplK gene, is transcribed during all stages of di¡erentiation and the transcription decreased in the course of di¡erentiation compared to vegetative substrate mycelium. Our results are also consistent with the analysis of the ribosomal protein pattern during the morphological di¡erentiation of S. antibioticus, where a decrease of several ribosomal proteins was observed in aerial mycelium, including a strong decrease in the L7/L12 content [2,4].
Acknowledgements We would like to thank Mrs. Rena¨ta Knirschova¨ for excellent technical assistance. This work was supported by a Grant 2/4007/98 from the Slovak Academy of Sciences.
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