Centro Nacional de Biotecnología, Campus de la Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
Correspondence
Rafael P. Mellado
rpmellado{at}cnb.uam.es
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ABSTRACT |
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The Streptomyces lividans ffh, scRNA and ftsY gene sequences described in this article have been deposited in GenBank under accession numbers AF071565, AY081854 and AY140960, respectively.
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INTRODUCTION |
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Major interactions among SRP components are evolutionarily conserved. Hence, Ffh contains two major domains. The NG domain, containing the GTP-binding site, structurally resembles the NG domain of the FtsY receptor and is involved in the interaction with the equivalent NG domain of the receptor. The Ffh C-terminal M domain is involved in the interaction with the nascent polypeptide chain and the scRNA. Both Ffh and FtsY present GTPase activity. The N-terminal A domain of the FtsY receptor contains the membrane-targeting signal and varies among FtsY homologues, probably in accordance with the different modes for targeting FtsY to the membrane (Powers & Walter, 1997).
The mammalian small size RNA (SRP RNA) is longer than 300 nt and contains two domains. The small domain (Alu domain) binds to SRP proteins 9 and 14 (Bui & Strub,1999). The Bacillus subtilis SRP system is similar to that of E. coli but its SRP RNA is larger (generally known as scRNA; 271 nt long) than in E. coli (generally known as 4·5S RNA; 114 nt long); it contains an Alu domain and the SRP harbours the B. subtilis histone-like (HBsu) protein as an additional component, interacting with the Alu domain of the scRNA (Nakamura et al., 1999
). The large domain (domain IV) of the small size RNA is fully conserved in mammalian and bacterial cells and is involved in the interaction with both the Ffh and the hydrophobic nascent polypeptide chain (Batey et al., 2000
). The interaction between Ffh and the 4·5S RNA represents the minimal functional SRP, all the bacterial SRP components being essential for cell growth.
Whereas in E. coli the SRP and the SecA/SecB-based secretion system(s) belong to two different targeting pathways functioning in a substrate-specific manner (Beck et al., 2000), a functional interaction has been detected between SecA and Ffh in B. subtilis (Bunai et al., 1996
). These differences between Gram-positive and Gram-negative bacteria may indicate the existence of different mechanisms for targeting secretory proteins. B. subtilis does not seem to have a SecB protein and the signal peptides of the B. subtilis secretory proteins seem to be more hydrophobic and longer than those of E. coli. Thus, the B. subtilis SRP system may be responsible for the targeting of both membrane and secretory proteins (Oguro et al., 1996
).
Gram-positive bacteria belonging to the genus Streptomyces are soil bacteria with mycelial growth that undergo a complex biochemical and morphological differentiation before forming exospore chains (Chater, 1998). Streptomycetes produce and secrete large quantities of proteins (Gilbert et al., 1995
) and Streptomyces lividans, in particular, has often been used as a host for the secretory production of heterologous proteins (Gilbert et al., 1995
; Van Mellaert & Anné, 1994
).
The genome sequence of Streptomyces coelicolor A3(2) (GenBank accession no. AL645882), an organism closely related to S. lividans, revealed the absence of a secB gene in this organism. The S. coelicolor genome is almost identical to that Cof S. lividans, from which secB appears to be missing as well (M. San Roman & R. P. Mellado, unpublished data), thereby rendering the study of the SRP complex very attractive. The identification and characterization of the S. lividans SRP system components are described in this study as a first step in exploring the possible role played by the S. lividans SRP in secretion.
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METHODS |
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Plasmid pUC19 (Norrander et al., 1983) was used to construct a BamHI library of gel-purified S. lividans TK21 chromosomal DNA fragments and to propagate cloned DNA sequences in E. coli. Plasmid pFF14 carries a 14 kb long BamHI chromosomal DNA fragment containing the ffh gene.
E. coli M15 is an E. coli K-12 derivative that harbours plasmid pREP4 (Villarejo & Zabin, 1974) and was purchased from Qiagen. The E. coli expression plasmid pQE30 (Qiagen) can provide a high level of expression of proteins carrying a hexahistidine (His6) tail at their amino ends. Plasmid pQE30 was used to propagate His6Ffh coding sequences in E. coli M15. Ampicillin (100 µg ml-1) and kanamycin (25 µg ml-1) were added to the media when needed.
DNA manipulation and PCR amplification.
General recombinant DNA manipulation was carried out as described previously (Hopwood et al., 1985; Sambrook et al., 1989
). Restriction endonucleases and DNA modifying enzymes were obtained from Boehringer Mannheim, Promega and Ecogene. S. lividans TK21 chromosomal DNA was used as a template for PCR amplification by incubation at 95 °C for 3 min, followed by 30 cycles of incubation at 95 °C (1 min), 45 °C (1 min) and 72 °C (2 min), with a final extension cycle at 72 °C for 10 min.
Expression and purification of His6Ffh for antibody preparation.
Oligonucleotides H15 (5'-CGCGGATCCACCGTCCGCTCTGTGCAG-3') and H16 (5'-CCCAAGCTTGCCCATGAACTTCTTGAA-3') were used as primers to amplify the Ffh coding sequences. The PCR-amplified DNA fragment was fused in-frame to the His6 coding sequences present in pQE30 under the control of the T5 promoter. The resulting plasmid, pQFH2, which encodes an Ffh protein tagged with a His6 tail at its amino end, was used to transform E. coli M15(pREP4).
The recombinant bacterium was grown at 37 °C with vigorous shaking and induced with 1 mM IPTG; the culture was allowed to grow for 5 h, as indicated in the QIAexpressionist manual (Qiagen). Ffh insoluble protein was recovered from a 500 ml culture of E. coli cells, as indicated in the QIAexpressionist manual. The cleared lysate supernatant containing the His6-tagged protein was loaded onto an equilibrated Ni-NTA spin column. The protein was extracted from the column by centrifugation as indicated in the QIAexpressionist manual. All buffers used were as described in the manual.
To raise polyclonal anti-Ffh antibodies in rabbits, purified Ffh preparations (50 µg in 500 µl) were mixed with 500 µl complete Freund's adjuvant and injected intramuscularly (2x1 ml) into a Hollander rabbit (Pfd : HOL) at intervals of 3 weeks. A blood sample was taken 2 weeks after applying the last injection, and the serum, collected by centrifugation (5 min, 150 g), was prepared as described by Dunbar & Schwoebel (1990). Polyclonal antibodies were obtained against agarase (DagA), as described previously (Parro & Mellado, 1994
).
Intracellular protein analysis and Western blot experiments.
Total intracellular proteins were visualized by Coomassie blue staining of 12·5 % SDS/polyacrylamide gels (Laemmli, 1970). For the Ffh Western blot analysis, intracellular proteins were separated by 12·5 % SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore) as described by Timmons & Dunbar (1990)
. Protein concentration in the different samples was determined as described by Bradford (1976)
using standard I bovine gamma globulin (Bio-Rad). The transferred material was incubated with antibodies raised against Ffh or DagA. Peptides reacting with the antibodies were revealed by incubation with 125I-labelled protein A [0·1 µCi ml-1 (3·7 kBq ml-1)] from Staphylococcus aureus (Amersham) as described by Timmons & Dunbar (1990)
. Membranes were exposed to Agfa Curix RP2 film at -70 °C.
Transcriptional analysis.
RNA was isolated as described previously (Parro et al., 1991) but with some modifications (Kedzierski & Porter, 1991
). Total RNA (30 µg) was transferred to nylon membranes (Hybond-N+; Amersham) and used for Northern blot analysis as described by Sambrook et al. (1989)
. Nylon membranes were incubated overnight at 65 °C in 0·5 M sodium phosphate pH 7·2/10 mM EDTA/7 % (w/v) SDS.
Oligonucleotides scRNA1 (5'-GCCCCCAACACGCTTTCGA-3') and scRNA2 (5'-GAGGTCGCGGCAGGACTGGC-3') were used to amplify a 493 bp long DNA fragment from the S. lividans chromosome containing the scRNA gene. The amplified DNA fragment (5 ng) was used as template to extend 10 pmol of primer scRNA4 (5'-TCCGGCCCTGGGGAGTTCAGT-3') with 5 U of Sequencing Grade Taq DNA polymerase (fmol DNA Cycle Sequencing System; Promega) in the presence of fmol DNA Sequencing 1xBuffer (Promega) and 10 µCi of [-32P]dCTP (10 µCi ml-1, 3000 Ci mmol-1; Amersham). The labelled DNA complementary to the scRNA was used as a specific probe for Northern blot analysis.
High-resolution S1 nuclease protection experiments were conducted as described previously (Sambrook et al., 1989; Barthelemy et al., 1986
; Parro et al., 1998
) using 50 µg total RNA. The DNA molecular size ladders were enzymically derived by the dideoxy chain termination method (Sanger et al., 1977
) by extension of primer scRNA4, 5'-labelled with 40 µCi [
-32P]ATP (10 µCi ml-1, 3000 Ci mmol-1; Amersham), using the 493 bp long DNA fragment as template. The radioactively labelled DNA fragment was also used to determine the transcription initiation site of the scRNA by providing protection from the S1 nuclease digestion. To determine the length of the scRNA, 100 µg total RNA were labelled with 50 µCi [
-32P]ATP (10 µCi ml-1, 3000 Ci mmol-1; Amersham) in the presence of T4 polynucleotide kinase (Sambrook et al., 1989
). The 493 bp long DNA fragment was used to determine the size of the 5'-endlabelled RNA by high-resolution S1 nuclease protection experiments.
RNA preparations were rendered DNA-free by incubation with DNase-RNase free (Promega) following the supplier's manual. The indicated amounts of total RNA preparations were used for the amplification reaction by incubation with AMV reverse transcriptase, followed by incubation with Thermus flavus DNA polymerase. The Promega Access RT-PCR system kit containing both enzymes was used for the RT-PCR analysis following the supplier's manual. RT-PCRs were carried out in a final volume of 100 µl using oligonucleotides H8 (5'-GATCGGTCAGGACGCGGTCAA-3') and H12 (5'-GAGCCGTTGATGATCGTCGG-3') as primers in the analysis of ffh transcription, and oligonucleotides ft4 (5'-GCACCTCGACGACGACACCTGGG-3') and ft5 (5'-GGATCAGACCGTTGTGGCCGGTGG-3') as primers in the analysis of ftsY transcription. The reverse transcriptase reactions were carried out in automated thermocyclers (PTC-100; MJ Research) by incubation at 48 °C for 45 min, followed by heating at 95 °C for 2 min. The amplification reactions were carried out by 40 cycles of incubation at 94 °C (0·5 min), 60 °C (1 min) and 68 °C (2 min), with a final extension step of incubation at 68 °C for 7 min. The amplified DNA fragments were analysed by 1·5 % (w/v) agarose gel electrophoresis.
Detection of scRNA associated with FfH and GTPase activity.
Bacteria from mid-exponential phase S. lividans cell cultures were harvested, concentrated four-fold in buffer I (50 mM triethanolamine pH 7·5/100 mM potassium acetate/1 mM EDTA/01 % (w/v) Triton X-100/1 mM DTT) and broken up with a French press [1000 p.s.i. (6·9 MPa)]. Aliquots (300 µl) from the cell lysate were pre-incubated at 4 °C for 5 min with 0·5 % (w/v) protein A-Sepharose CL-4B (Sigma). The cleared lysate was incubated for 12 h with anti-Ffh serum at 4 °C before adding protein A-Sepharose again, and incubation continued for a further 2 h. Immunoprecipitated material was collected by centrifugation and recovered in 20 µl buffer I.
The scRNA contained in the immunoprecipitated material was recovered by heating to 65 °C in 1 : 1 mixtures of 4 M guanine thiocyanate/phenol. The denatured RNA was analysed by Northern blot.
The Ffh protein present in the immunoprecipitate was visualized by Western blot analysis using anti-Ffh serum. To determine the GTPase activity associated with the immunoprecipitated Ffh, half of the immunoprecipitated material was incubated at 37 °C for 1 h with 100 µCi [-32P]GTP (10 mCi ml-1, 3000 Ci mmol-1; Amersham) in a final volume of 20 µl of 20 mM Tris/HCl pH 7·5/2 mM MgCl2/10 % (w/v) glycerol/0·1 mg ml-1 BSA/1mM GTP. One-tenth of the reaction (equivalent to 15 µl cell lysate) was applied to a PEI-cellulose sheet (Aldrich) and developed with 0·5 M potassium phosphate pH 3·5. The radioactively labelled GDP released by the GTPase hydrolytic action was revealed by exposure to Agfa Curix RP2 film at -70 °C.
Co-immunoprecipitation of FfH and pre-DagA.
Aliquots (300 µl) from French press lysates of S. lividans TK21Y62(pAGAs5) cell cultures harvested at the transition to the stationary phase of growth were immunoprecipitated by incubation with anti-Ffh serum or anti-DagA serum, as described above. The presence of pre-agarase in the anti-Ffh immunoprecipitated material or Ffh in the anti-DgaA immunoprecipitate was determined by Western blot analysis using serum anti-DagA or anti-Ffh, respectively.
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RESULTS |
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Transcriptional analysis of the SRP components
Submerged cultures of S. lividans TK21 were incubated at 30 °C in NMMP medium supplemented with 0·5 % (w/v) mannitol as carbon source. They grew exponentially with a doubling time of 4·2 h. The transition to stationary phase occurred 2530 h after inoculation, at biomass dry weights of
2·5 mg ml-1. Transcriptional analysis of the ftsY gene by Northern blot confirmed that ftsY is transcribed as a single gene transcriptional unit, and the ftsY transcript was detected throughout the different phases of bacterial cell culture growth (Fig. 3
a), apparently being more abundant at the early-exponential phase of growth, as confirmed by RT-PCR analysis (not shown).
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The ffh gene is likely to form part of an operon containing at least two genes, as deduced from the sequencing data. RT-PCR analysis using oligonucleotides H8 and H12 as primers revealed that the ffh transcript was present throughout the different phases of bacterial cell culture growth (Fig. 3c), the transcript being considerably more stable than the glnD transcript (not shown), suggesting a differential post-transcriptional processing of the glnD and ffh messengers. A putative downstream box that is complementary to the 16S rRNA (Wu & Janssen, 1996
; Sprengart et al., 1996
) was identified in the ffh transcript, which may ensure its translation. Table 1
shows the putative downstream boxes of ffh and signal peptidase (sipW and sipZ) transcripts (Parro et al., 1999
) compared to that of the viomycin phosphotransferase (vph) transcript (Wu & Janssen, 1996
) and the S. lividans 16S rRNA complementary sequence.
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Anti-Ffh antibodies were used to immunoprecipitate S. lividans TK21 cell lysates. The presence of Ffh in the resulting immunoprecipitated material was further analysed by Western blot experiments and its capacity to hydrolyse GTP was analysed by TLC. The presence of accompanying scRNA in the immunoprecipitate was analysed by Northern blot experiments. A unique protein of the expected molecular size was detected reacting with the anti-Ffh serum (Fig. 4a); GTPase activity was also present in the immunoprecipitate (Fig. 4b
) and an scRNA of the expected mobility was detected as well (Fig. 4c
).
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SRP is involved in protein secretion
S. lividans TK21Y62(pAGAs5) is an S. lividans TK21 mutant strain in which the major signal peptidase (SipY) is inactive and which carries the S. coelicolor agarase gene dagA on the multicopy plasmid pAGAs5 (Palacín et al., 2002). This strain is able to overproduce agarase, although pre-agarase processing takes place at a much lower speed than in S. lividans TK21(pAGAs5) and, as a result, pre-agarase tends to accumulate intracellularly at a higher level. To determine if this minimal functional SRP was involved in targeting secretory proteins, S. lividans TK21Y62(pAGAs5) cell lysates were immunoprecipitated with anti-Ffh antibodies or anti-agarase antibodies. The presence of either agarase or Ffh in the immunoprecipitated material was analysed by Western blot using the corresponding antiserum in each case. Anti-agarase antibodies precipitated the pre-agarase present inside the cell (Fig. 5
a, lane 1), whereas anti-Ffh antibodies only precipitated the pre-agarase bound to the Ffh (Fig. 5a
, lane 2). Anti-Ffh antibodies precipitated the Ffh present inside the cell (Fig. 5b
, lane 2), whereas anti-agarase antibodies only precipitated a comparatively small amount of Ffh bound to pre-agarase (Fig. 5b
, lane 1). Differences in relative band intensity, as revealed by Western blot analysis, could also reflect differences in the relative specificity of each antibody. The fact that both antibodies co-immunoprecipitated the other protein indicates that an interaction of Ffh and pre-agarase does occur inside the cell, therefore suggesting that the SRP system is involved in the S. lividans protein secretion pathway.
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DISCUSSION |
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The S. lividans SRP system consists of a functional SRP, a ribonucleoprotein, composed of Ffh, and an 82 nt long RNA. The small size of this RNA, as occurs in E. coli, seems to leave no room for other proteins to adhere to the complex, although at present this cannot be totally ruled out. Fig. 6 shows a comparison of the E. coli, B. subtilis and S. lividans SRP structures. FtsY, the receptor protein, also forms part of the S. lividans SRP system and its expression takes place throughout the cellular growth similar to that of the scRNA and Ffh proteins. So far, all attempts made by us to obtain mutants in any of the three corresponding genes have failed, thus indicating the possible essential nature of the SRP components in S. lividans. Interestingly enough, transcription of S. lividans signal peptidase genes seems to occur in the shape of polycistronic messengers that are specifically processed (Parro et al., 1999
), as appears to be the case for ffh transcription. The ffh transcript seems to contain an internal downstream box (Wu & Janssen, 1996
) to ensure translation of the processed messenger, a feature also shared with the sip transcripts (Table 1
; Parro et al., 1999
).
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The construction of a His6-tagged FtsY protein, currently under way, will help in the design of experiments to elucidate the role played by the FtsY receptor in SRP-mediated secretion in S. lividans.
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ACKNOWLEDGEMENTS |
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Received 20 February 2003;
revised 23 May 2003;
accepted 23 May 2003.
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