Four genes encoding different type I signal peptidases are organized in a cluster in Streptomyces lividans TK21

Víctor Parro1, Sabine Schacht2, Jozef Anné2 and Rafael P. Mellado1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21. Three of the sip genes constitute an operon and the fourth is the first gene of another operon encompassing three additional, unrelated genes. A DNA fragment containing the four sip genes complemented an Escherichia coli type I signal peptidase mutant when cloned in a multicopy plasmid. Clustering of four different type I signal peptidase genes seems, so far, to be a unique feature of Streptomyces.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Proteins to be exported are typically synthesized as preproteins with an amino-terminal peptide that destines the protein for the secretion pathway. The signal peptide is needed both to target the protein and to initiate translocation across the membrane (von Heijne, 1990 ; Wickner et al., 1991 ; Pugsley, 1993 ; Driessen, 1994 ). Upon translocation, the preproteins have their signal peptides removed by the action of signal peptidases (SPases). Prokaryotic type I SPases, also known as leader peptidases (Lep), process the majority of exported preproteins. Homologues of type I SPases have been identified in the endoplasmic reticulum and mitochondria of eukaryotic cells and, together with the prokaryotic type I SPases, they appear to belong to a novel family of serine proteases (Dalbey & von Heijne, 1992 ; Dalbey et al., 1997 ;van Dijl et al., 1992 , 1995 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
Escherichia coli K514 was used as a host for library construction and was cultivated at 37 °C in LB medium (Murray, 1983 ) containing 100 µg ampicillin ml-1. S. lividans TK21 was cultivated in YEME or NMMP media at 30 °C (Hopwood et al., 1985 ). Plasmid pRM1 (Parro et al., 1991 ) was used to construct the library in E. coli. The multicopy plasmid pIJ486 (Ward et al., 1986 ) and the E. coli–S. lividans shuttle plasmid pUWL218 (Wehmeier, 1995 ) were used to propagate the sip genes in S. lividans. E. coli IT89 is a tetracycline-resistant derivative of E. coli KH5402 tyr(Am) trpE9829(Am) thr metE ilv thy supF6 (ts) (Osawa & Yura, 1980 ) in which the mutation lep-9 (type I SPase ts) has been introduced by P1 transduction (Inada et al., 1989 ). E. coli IT89 was used to propagate the shuttle plasmid pUWL218 at 32 °C in LB medium containing 100 µg ampicillin ml-1 and 5 µg tetracycline ml-1 for selection and maintenance of transformants. Plasmid pIJ486 was used to propagate sip promoters in S. lividans.

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 [{alpha}-32P]dCTP was used instead of [{alpha}-32P]dATP. Probes for S1 nuclease mapping are indicated in Fig. 1(a) and comprised the following fragments: NcoI1–NcoI2 (1846 bp, probe 1); AlwNI1–BamHI1 (5673 bp, probe 2) and SalI4–BbrPI2 (1721 bp, probe 3), labelled at their respective NcoI2, AlwNI1 and BbrPI2 5' ends for low-resolution S1 nuclease experiments. Fragments NcoI1–SalI1 (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 AvaII1–sn29 (186 bp, probe 7), TaqI–sn28 (5'-TGCGCTGGAGTTTGCGGCGCTCGG-3') (239 bp, probe 8), NcoI3–PstI3 (730 bp, probe 9), Sau3AI1–Sau3AI2 (411 bp, probe 10), Sau3AI3–Sau3AI4 (205 bp, probe 11) and AflIII–NcoI5 (361 bp, probe 12), labelled at their respective AvaII1, TaqI, NcoI3, Sau3AI1, Sau3AI3 and AflIII 3' ends.



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Fig. 1. Restriction and transcriptional maps of cloned DNA containing the four sip genes. (a) Restriction map of the 7860 bp BamHI fragment cloned from S. lividans TK21 chromosome. Only relevant restriction endonuclease sites are indicated: Av, AvaII; Af, AflIII; Aw, AlwNI; B, BamHI; Bb, BbrPI; Bg, BglII; K, KpnI; Nc, NcoI; P, PstI; Sl, SalI; Sa, Sau3AI; Sp, SphI; Tq, TaqI; Xh, XhoI. The numbers identify different cleavage sites for the same restriction endonuclease. The various DNA probes used in the nuclease S1 mapping experiments are numbered from 1 to 12 and are described in more detail in Methods. Probes are labelled at their 5' (*) or 3' ({bullet}) ends. (b) Transcriptional map of the sip region. Large arrows indicate the direction and extent of transcription. Numbers 1–4 indicate the ORFs of unidentified genes.

 
Total RNA transferred to nylon membranes (Hybond-N+; Amersham) was used for Northern analysis as described by Sambrook et al. (1989 ). Nylon membranes were incubated overnight at 52 °C in the presence of 80% (v/v) formamide, 1·7% (w/v) SDS, 25 mM EDTA and 0·1 M sodium phosphate pH 7·0.

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 [{gamma}-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and determining the primary structure of sip genes
From the conserved amino acid regions of known type I SPases (van Dijl et al., 1992 ), two oligonucleotides were designed for PCR amplification of sequences from genomic S. lividans TK21 DNA. The regions chosen were G89–L95 (box B) and M271–N277 (box E). The corresponding degenerate oligonucleotides sn1 [5'-GG(G / C)TC(G / C)ATGATGCC(G / C)AC(G / C)CT(G /C)-3'] and sn3 [5'-GTT(G/C)CG(G/C)CGGTTGTC(G/C)CCCAT-3'] were synthesized, taking into account the Streptomyces codon bias (Wright & Bibb, 1992 ). BamHI sites were added to the 5' end of each oligonucleotide primer to be used for cloning the amplified DNA. A 310 bp DNA fragment was amplified, cleaved with BamHI and inserted into plasmid pRM1. Both strands of the insert were sequenced, and the deduced amino acid sequence was compared with known SPase sequences. Regions matching conserved sequences (R68–V73, V82–R84 and N103–G104) in the SipS sequence (van Dijl et al., 1992 ) were apparent.

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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Table 1. ORF features

 
Signal peptidases W, X, Y and Z
The deduced amino acid sequences for SipW, SipX, SipY and SipZ showed extensive similarity to type I SPases of both Gram-positive and Gram-negative bacteria, as well as to SPases of eukaryotic origin. Fig. 2 shows an alignment of the four SPases with the SipS SPase from Bacillus subtilis. All the amino acids considered relevant for the activity of bacterial SPases (Black, 1993 ; Tschantz et al., 1993 ; van Dijl et al., 1995 ) are conserved in the four S. lividans Sip proteins. In particular, the serine and lysine residues at positions 43 and 83 of the B. subtilis SipS, which are thought to be needed for SPase catalytic activity, are included in the conserved amino acid regions (boxes B and D, respectively, of the four S. lividans SPase sequences). The other conserved domains (boxes A, C and E) are also present.



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Fig. 2. Amino acid sequence alignments of the four S. lividans SPases and SipS from B. subtilis. Boxes A, B, C, D and E indicate the conserved domains previously identified for type I SPases (van Dijl et al., 1992 ). Residues needed for the activity or the stability of SipS (van Dijl et al., 1995 ) are indicated in bold face. The putative transmembrane anchors at the carboxyl end are underlined.

 
The von Heijne algorithms (von Heijne, 1992 ; Sipos & von Heijne, 1993 ) identified a single N-terminal membrane anchor domain at conserved box A in the Gram-positive type I SPases (Tjalsma et al., 1997 ). This contrasts with most SPases of Gram-negative bacteria, which have two N-terminal transmembrane anchors (van Dijl et al., 1992 ). In addition, a carboxy-terminal membrane-spanning region was predicted for SipX, SipY and SipZ (underlined in Fig. 2), a feature also reported in the Rhodococcus capsulatus SPase (database accession number Z68305) and in the B. subtilis SipW (Tjalsma et al., 1998 ). The presence of this C-terminal transmembrane anchor should, however, have no effect on the extracellular positioning of the conserved serine–lysine catalytic dyad.

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 sipW–sipX–sipY operon.

To study the transcriptional organization of the region, a 6439 bp long BglII–BamHI 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 25–30 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 sipW–sipX 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 BamHI1–BamHI2 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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Fig. 3. Low-resolution S1 nuclease mapping. Lane 1, 50 µg E. coli MRE600 tRNA (Boehringer Mannheim); lane 2, 50 µg total RNA from S. lividans TK21(pSNW23) culture extracted 17 h after inoculation. The probes were 32P-labelled DNA fragments comprising regions sipW–sipX (a), rplS–sipY (b) and sipZ–mutT (c). The sizes of the protected fragments in nucleotides are indicated.

 
To ascertain if the transcriptional pattern observed was due to genuine initiation events, the different DNA regions containing the putative promoters were individually cloned in the promoter-probe vector pIJ486 containing a promoterless kanamycin resistance gene, propagated in S. lividans and tested for their ability to confer kanamycin resistance on the cells harbouring them. The only putative promoters confirmed were those in front of the rplS, sipW and sipZ genes, leaving sipX, sipY and mutT with no effective promoter in front of them. Putative transcription initiation sites were determined by high-resolution S1 nuclease protection experiments, with total RNA from the S. lividans TK21(pUWL218) or S. lividans TK21(pSNW23) cultures. Transcription initiating at rplS, sipW and sipZ promoters was mapped using the single-stranded probes 4, 5 and 6 (Fig. 1), respectively, SalI1 (23 nt downstream from rplS ATG translation start codon), sn29 (151 nt downstream from sipW ATG translation start codon) and sn39 (53 nt downstream from sipZ ATG translation start codon). Fig. 4 shows the transcription initiation sites for the confirmed promoters. Protected fragments 158 nt long, 156 nt long (corresponding to the 1020 nt long one in Fig. 3a) and 58 nt long (corresponding to the 1200 nt long one in Fig. 3c) were identified for transcription initiating in front of rplS, sipW and sipZ, respectively.



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Fig. 4. High-resolution S1 nuclease mapping. Transcription initiation sites at rplS (a), sipW (b) and sipZ (c) were determined using 50 µg total RNA from S. lividans (pUWL218) (lanes 1–4) and S. lividans (pSNW23) (lanes 5–8) cultures extracted at 17 (lanes 1, 5), 22 (lanes 2, 6), 38 (lanes 3, 7) and 45 (lanes 4, 8) h after inoculation. Lanes R and Y contain the purine and pyrimidine sequence ladders, respectively, resulting from chemical degradation of the corresponding radioactively labelled probes run in parallel. Arrows indicate the size of the protected fragment in nucleotides.

 
The level of transcription seemed to be rather low when the sip genes were present in single copy, increasing generally when the genes were propagated in multicopy. The two mapped sip promoters seemed to initiate transcription more efficiently in late phases of growth when the genes were cloned in the multicopy plasmid, whereas rplS transcription was more abundant in the early phases of growth (Fig. 4a). The -10 and -35 regions for the sip promoters are compared with the consensus sequence for the Streptomyces major RNA polymerase promoters in Table 2. The sipW and sipZ transcripts belong to a class of transcripts lacking a classical RBS and having instead a so-called downstream box within their respective sequences that is complementary to the 16S rRNA (Wu & Janssen, 1996 ; Sprengart et al., 1996 ). The putative downstream boxes of sipW and sipZ transcripts compared to that of the viomycin phosphotransferase gene (vph) transcript and the S. lividans 16S rRNA complementary sequence (Wu & Janssen, 1996 ) are shown in Table 3.


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Table 2. Mapped promoters

 

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Table 3. Putative downstream boxes for sipW and sipZ compared to that of vph

 
Transcription termination sites were also mapped by S1 nuclease protection experiments. Transcription terminating behind rplS, sipW, sipX, sipY, sipZ and mutT was mapped using the single-stranded probes 7, 8, 9, 10, 11 and 12 (Fig. 1), respectively, labelled at their 3' ends. Protected fragments of 72–76 and 182–186 nt were identified for transcription terminating behind rplS and sipY, respectively (Fig. 5). No transcription termination signals were found behind sipW, sipX, sipZ and mutT, as expected. The predicted secondary structures for the putative terminators are also shown in Fig. 5. Fig. 1b is a transcriptional map of the region.



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Fig. 5. Transcription termination. Transcription termination sites for rplS (a, lane 1) and sipY (c, lane 1) were determined by S1 nuclease mapping using 50 µg total RNA from S. lividans (pSNW23) cultures extracted 17 h (rplS) or 38 h (sipY) after inoculation. The size in nt of the protected fragments is indicated. Lanes R and Y contain the purine and pyrimidine sequence ladders respectively, resulting from chemical degradation of the corresponding radioactively labelled probes run in parallel. Stem–loop structures for the rplS (b) and sipY (d) terminators are also shown. Arrows point to the nucleotides where transcription ended.

 
Complementation of the SPase type I deficiency in E. coli
E. coli IT89 carries a ts mutation in the only type I SPase gene that it harbours. To check if this deficiency could be complemented by the S. lividans sip genes, E. coli IT89(pSNW23) and E. coli IT89(pUWL218) carrying plasmids containing the four signal peptidase genes or the vector, respectively, were cultivated in LB at 32 °C until they reached exponential phase (OD600 0·3). Each culture was then divided into two by dilution to an OD600 of 0·08 with pre-warmed medium at 32 or 42 °C, respectively, and incubation was continued at both temperatures. All cultures grew well at 32 °C, whereas only the cells carrying the recombinant plasmid were able to grow at the restrictive temperature, as expected if one or several of the S. lividans sip genes could complement the E. coli lep-9 mutation. In preliminary experiments, neither SipW nor SipZ seemed to complement the E. coli lep-9 mutation when their respective genes were individually cloned in a multicopy plasmid and propagated in E. coli IT89 (not shown). Cloning of sipX or sipY on multicopy plasmids resulted in recombinant cells that were difficult to culture and prone to cell lysis when they were used to inoculate liquid medium. More experiments are needed to ascertain which sip gene(s) is/are responsible for complementing the E. coli lep-9 mutation.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Derived amino acid sequences of at least 20 different type I SPases are available from recent DNA sequencing data. As in E. coli, most genomes contain only one type I SPase gene (Blattner et al., 1997 ). However, B. subtilis, Bacillus amyloliquefaciens and Staphylococcus aureus (Cregg et al., 1996 ) are exceptions. B. subtilis contains five different chromosomal type I SPase genes (sipS, sipT, sipV, sipU and sipW; Kunst et al., 1997 ; Tjalsma et al., 1998 ) plus two plasmid-encoded SPases (Meijer et al., 1995 ). Furthermore, two type I SPases have been described for B. amyloliquefaciens (see Meijer et al., 1995 ). In this paper we show that four type I SPase genes are clustered in the S. lividans genome. The same arrangement of four sip genes has recently been found in the S. coelicolor genome sequence (database accession number AL023797) and in Streptomyces venezuelae (S. Schacht, V. Parro, R. P. Mellado & J. Anné, unpublished results), whereas in B. subtilis the SPase genes are scattered over the genome. This so far unique feature of SPase gene clustering in streptomycetes leads us to presume that expression of the four sip genes may be subject to specific regulation.

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 (27–30% 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.


   ACKNOWLEDGEMENTS
 
This research was supported by Grants BIO94-0792 and BIO97-0650-C02-01 from the Spanish CICYT and by the European Union Grant BIO4-98-0051. We thank Julio Gutierrez for the E. coli IT89 strain. We are grateful to Sierd Bron for useful discussions on SPases. Sabine Schacht is a fellow of IWT (Flemish Institute for the Promotion of Scientific-Technological Research in Industry).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 January 1999; revised 30 March 1999; accepted 4 May 1999.