Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium2
Author for correspondence: Rafael P. Mellado. Tel: +34 91 5854547. Fax: +34 91 5854506. e-mail: rpmellado{at}cnb.uam.es
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ABSTRACT |
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Keywords: signal peptidases, secretion, Streptomyces
Abbreviations: SPase(s), signal peptidase(s)
The GenBank accession number for the sequence data reported in this paper is Z86111.
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INTRODUCTION |
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Most Gram-negative bacterial type I SPases contain two amino-terminal transmembrane segments, and have their active site in their carboxyl-terminal parts exposed to the periplasm (Dalbey & von Heijne, 1992 ). Type I SPases from Gram-positive bacteria share a higher degree of homology than do the SPases of Gram-negative bacteria: they are much smaller and have only one amino-terminal anchor domain (Meijer et al., 1995
; Hoang & Hofemeister, 1995
).
Streptomycetes are Gram-positive bacteria known to secrete large quantities of proteins (Gilbert et al., 1995 ). The mechanism of protein secretion is not well characterized, but signal-dependent protein secretion would be expected to use components organized like those of other bacteria. Secretion signals of proteins secreted from streptomycetes have been used to obtain secretion of heterologous proteins (Brawner et al., 1991
; Anné & van Mellaert, 1993
). This paper reports the identification, cloning, sequencing and expression of a cluster of four adjacent type I SPase genes from Streptomyces lividans. The DNA sequences of two of these genes have been reported previously (Parro & Mellado, 1998
; Schacht et al., 1998
). The clustering of four SPase genes in S. lividans and their co-transcriptional control makes this species unique among Gram-positive bacteria with multiple type I SPase genes.
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METHODS |
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DNA manipulation, PCR amplification and colony hybridization.
DNA was manipulated as described by Sambrook et al. (1989 ) and Hopwood et al. (1985
). Restriction endonucleases and DNA modifying enzymes were obtained from Boehringer Mannheim, Promega and Ecogene. DNA fragments were purified from low-melting-point agarose gels (LM3; Hispanagar) using an agarase from Streptomyces coelicolor overproduced and purified in our laboratory (Parro et al., 1997
). S. lividans TK21 genomic DNA was used as a template for PCR amplification. Denaturing at 95 °C for 3 min was followed by 30 cycles of 95 °C for 1 min, 45 °C for 1 min and 72 °C for 2 min. The amplification ended with a 10 min incubation at 72 °C. Preparations of total RNA from bacterial cultures and S1 nuclease protection experiments were done as described previously (Parro & Mellado, 1993
, 1998
), except that for labelling of the TaqI 3' ends [
-32P]dCTP was used instead of [
-32P]dATP. Probes for S1 nuclease mapping are indicated in Fig. 1(a
) and comprised the following fragments: NcoI1NcoI2 (1846 bp, probe 1); AlwNI1BamHI1 (5673 bp, probe 2) and SalI4BbrPI2 (1721 bp, probe 3), labelled at their respective NcoI2, AlwNI1 and BbrPI2 5' ends for low-resolution S1 nuclease experiments. Fragments NcoI1SalI1 (447 bp, probe 4), sn30 (5'-CTGCGCGAGCTGCGCGGCAAGGC-3')sn29 (5'-TCACGGTGATCCGGCCGCCGGGC-3') (253 bp, probe 5) and sn10 (5'-CCCTGCCGGTCCCCGACACC-3')sn39 (5'-CCCTTGTTCGCTCCGCCGCCGCGC-3') (317 bp, probe 6), were labelled at their respective 5' ends for high-resolution S1 nuclease experiments. Probes for S1 nuclease mapping of transcription termination sites are also indicated in Fig. 1(a
) and comprised fragments AvaII1sn29 (186 bp, probe 7), TaqIsn28 (5'-TGCGCTGGAGTTTGCGGCGCTCGG-3') (239 bp, probe 8), NcoI3PstI3 (730 bp, probe 9), Sau3AI1Sau3AI2 (411 bp, probe 10), Sau3AI3Sau3AI4 (205 bp, probe 11) and AflIIINcoI5 (361 bp, probe 12), labelled at their respective AvaII1, TaqI, NcoI3, Sau3AI1, Sau3AI3 and AflIII 3' ends.
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Colonies from a genomic DNA library of S. lividans TK21 were transferred to nylon filters (Hybond-N+; Amersham) and screened for hybridization to the appropriate DNA fragment obtained by PCR amplification from the S. lividans chromosome and labelled at its 5' end with 32P.
DNA sequencing and computer analysis of sequences.
Plasmid DNA was sequenced by the chemical degradation method (Maxam & Gilbert, 1980 ) and by the dideoxy chain-termination method (Sanger et al., 1977
) using the fmol kit from Promega and synthetic oligonucleotides 5'-labelled with [
-32P]ATP (Amersham). For automated sequencing an Applied Biosystems 373 DNA sequencer was used.
The DNA sequence was analysed for ORFs using codonpreference (UWGCG), with a Streptomyces codon usage table based on 64 sequenced genes (Wright & Bibb, 1992 ). fasta and pileup programs were also used to search databases for sequence similarities and to align sequences with known SPases.
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RESULTS |
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Genomic BamHI fragments were ligated into pRM1 and used to transform E. coli K514. When the genomic library so constructed was screened for hybridization to the 310 bp PCR-amplified fragment, only 2 of 2700 transformants examined gave a positive result. One of them harboured the recombinant plasmid pSN40 carrying a 7860 bp fragment that included the 310 bp probe sequence. The sequence of a 909 bp subfragment from this clone, encompassing the gene sipZ, was obtained by using synthetic oligonucleotides to walk along the cloned DNA. Walking upstream and downstream of sipZ gave the complete sequence of the cloned DNA fragment, which included 3 additional sip genes. Thus a total of 12 genes, encoding 4 different SPases, flanked by 8 unrelated genes were identified (Fig. 1b). The amino acid sequence of the putative product of each ORF was compared with the non-redundant protein databases (Table 1
).
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Transcription of the sip genes in S. lividans
The nucleotide sequence of the cloned DNA fragment indicated that sipW has an 8 bp overlap with sipX, whilst the sipX stop codon is only 9 bp upstream of the sipY start codon, suggesting that sipW, sipX and sipY could be organized as an operon. The sipZ coding sequence is separated from that of sipY by 104 bp, within which a rho-independent transcription terminator was identified. Therefore, sipZ transcription could be regulated independently from that of the putative sipWsipXsipY operon.
To study the transcriptional organization of the region, a 6439 bp long BglIIBamHI subfragment containing the four sip genes and other adjacent genes was transferred to the multicopy plasmid pUWL218, generating plasmid pSNW23 for propagation in S. lividans TK21. Submerged cultures of S. lividans TK21(pUWL218) or S. lividans TK21(pSNW23) were incubated at 30 °C in NMMP medium supplemented with 0·5% (w/v) mannitol as carbon source. They grew exponentially with doubling time of approximately 4·2 h. The transition to stationary phase occurred 2530 h after inoculation, at biomass dry weights of approximately 2·5 mg ml-1.
Low-resolution S1 mapping experiments with total RNA from mid-exponential phase S. lividans TK21(pSNW23) cultures detected several putative transcripts in the DNA region containing the four sip genes (Fig. 3). Probes 1, 2 and 3 (Fig. 1
) detected protected fragments corresponding to transcripts covering most of the sipWsipX region (transcripts 1020, 980, 940, 740, 720 and 640 nt long; Fig. 3a
); protected fragments corresponding to a 2300 nt long transcript covering half of sipW, all sipX and most of sipY and to two transcripts (1050 and 990 nt long) covering only sipY (Fig. 3b
); and a protected fragment corresponding to a 1200 nt long transcript covering all sipZ and most of mutT (Fig. 3c
), respectively. These results suggest that transcription from the sipW promoter may continue through sipX and sipY, and that transcription started at sipZ may continue through mutT. A 2000 nt long transcript was also detected when the 7860 bp long BamHI1BamHI2 fragment (see Fig. 1a
) labelled at its BamHI2 5' end was used as a probe (not shown), indicating that transcription starting at sipZ could continue to the end of the cloned DNA. The detection of different transcripts by S1 nuclease mapping suggests the existence of many transcription initiation sites in the region or, alternatively, the existence of selective mRNA processing as a mechanism regulating the presence of sip messengers.
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DISCUSSION |
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The sequencing data and the transcriptional analyses suggest that sipW, sipX and sipY are transcribed as a polycistronic mRNA. Transcription under the control of sipW and sipZ promoters starts close to the ATG translation initiation codon, a feature that has been described for Streptomyces and E. coli genes having a translational downstream box that acts as an efficient translation initiation signal when a classical RBS is missing (Sprengart et al., 1996 ). Both sipW and sipZ mRNAs have such a downstream box (Table 3
). Two well-defined transcription termination signals were detected, one downstream of rplS, which leaves the sipW promoter as the first possible promoter for a sip operon, and the other right after sipY; no signals for termination of transcription initiated at the sipZ promoter were detected within the cloned DNA.
Sequences around the 5' ends of the transcripts detected by low-resolution S1 nuclease mapping and to which no promoter could be assigned did have a higher A+U content than predicted from the normal base composition of Streptomyces mRNA (2730% A+U). These 5' ends may result from the processing of larger mRNAs, as an A+U rich content has been associated with specific RNase E cleavage in E. coli (McDowall et al., 1994 ). The possible existence of specific mRNA processing, by an RNase E analogue or another factor, might be a potential way to regulate the relative amount of some sip messengers, as described for the E. coli cell division genes ftsA and ftsZ mRNA (Cam et al., 1996
; Flärdh et al., 1997
). A direct repeat, CCGGAGCGGCGCCCGAC, separated by a C residue appears 10 nt in front of the sipX translation initiation codon; another direct repeat, GGAACTCCCG, separated by 8 residues appears 66 nt upstream of the sipY translation initiation codon. The role played by these direct repeats, if any, remains to be elucidated, but they may represent potential sites for regulatory events that may account for some of the apparently specific processing of RNA detected. The 6 nt long direct repeat (CCGGGG) and the 10 nt long inverted repeat (CGGGGCCGGA, which forms a part of the rplS transcription terminator) located around the -35 and -10 regions of the sipW promoter may also be associated with as yet unknown regulatory events.
Complementation of the E. coli lep-9 mutation indicates that at least one of the S. lividans Sip proteins functions in E. coli, strongly suggesting that one of these sip genes encodes a polypeptide with type I SPase activity.
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ACKNOWLEDGEMENTS |
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Received 5 January 1999;
revised 30 March 1999;
accepted 4 May 1999.