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Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
Correspondence
Günther Muth
gmuth{at}biotech.uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Institute of Molecular Biology and Biophysics, ETH Zurich, HPK D14.1, Schafmattstrasse 20, 8093 Zurich, Switzerland. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast to most other bacteria, streptomycetes do not divide by binary fission, but grow by apical tip extension (Flardh, 2003b). The hyphal filaments septate infrequently, forming a branched substrate mycelium containing multiple nucleoids (Flardh, 2003a). In response to nutrient limitation, aerial hyphae grow up from the substrate mycelium, and become synchronously septated to form uninucleoid spore chains (Hopwood, 2006).
The substrate mycelium of Streptomyces has been shown to be capable of exchanging DNA by conjugation (Kieser et al., 1982; Hopwood & Wright, 1973). A single plasmid-encoded protein (TraB) is sufficient to promote intermycelial plasmid transfer from the donor into the recipient (Kieser et al., 1982; Maas et al., 1998; Kosono et al., 1996; Pettis & Cohen, 1994). TraB belongs to the septal DNA translocator proteins of the FtsK family, and has been shown to recognize the clt region, which is a specific sequence of approximately 50 bp that is required for plasmid transfer (Reuther et al., 2006a). Binding of TraB to the clt region does not involve processing of the DNA, suggesting that double-stranded plasmid DNA is translocated during Streptomyces conjugation. Localization of TraB to the hyphal tip has indicated that Streptomyces conjugation proceeds at the hyphal tip (Reuther et al., 2006a).
In adaptation to the mycelial growth characteristics of Streptomyces, primary plasmid transfer at the hyphal tips is followed by a series of secondary transfer processes within the recipient mycelium (Hopwood & Kieser, 1993). The newly transferred plasmid is most probably translocated via the septal cross-walls to the neighbouring mycelial compartments, although this model needs to be confirmed by experimental data. Plasmid spreading results in the rapid colonization of the recipient mycelium with the plasmid. Because unregulated expression of the transfer and spread of genes is detrimental (Kendall & Cohen, 1987; Pettis et al., 2001), the conjugation process is associated with temporary retardation of growth and morphological differentiation. When a plasmid-carrying Streptomyces spore germinates on a lawn of plasmid-free recipients, inhibition zones (pocks) of 1–3 mm are formed. These pock structures indicate the area where the recipient mycelium has obtained a plasmid (Hopwood & Kieser, 1993).
Whereas the primary transfer from the donor to the recipient requires only TraB (Pettis & Cohen, 1994), several plasmid-encoded spread (Spd) proteins are involved in intramycelial plasmid spreading via the septal cross-walls (Kataoka et al., 1991; Servín-González et al., 1995). The spd genes of the different plasmids are often cotranscribed, and overlap in their stop and start codons. They encode hydrophobic proteins of different sizes, some of which are very small (50–100 aa), and they do not show any sequence conservation or similarity to any other proteins in databases (Grohmann et al., 2003). Therefore, the molecular function of the Spd proteins is completely unknown.
Plasmid pSVH1 is a 12 652 bp conjugative pock-forming plasmid from Streptomyces venezuelae. pSVH1 contains two operons of translationally coupled genes, where insertional mutagenesis has shown involvement in pock formation (Reuther et al., 2006b). We report the characterization of the pSVH1 spdB3–spd79–spdB2 operon, and show that SpdB2 is an oligomeric integral membrane protein that binds dsDNA, and interacts with Spd79 and TraB. This indicates that a complex DNA-translocation apparatus is inserted into the septal cross-walls to promote intramycelial plasmid spreading during Streptomyces conjugation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), E. coli BL21(DE3)pLys (Invitrogen) and Streptomyces lividans TK64 (Hopwood et al., 1983) were used. E. coli and S. lividans strains were cultivated as described (Sambrook, 2001; Kieser et al., 2000). Plasmids and primers used in this study are listed in Tables 1 and 2, respectively.
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, underlined nucleotides) for NdeI and HindIII, and a His6-encoding sequence was incorporated at the C-terminal end of spdB2. The resulting product was cloned into pJoe2775 that had been cut with NdeI and HindIII (J. Altenbuchner, personal communication), and this resulted in pBG1. For expression in S. lividans, the PCR fragment described above was subcloned into pDrive, and inserted as a NdeI–EcoRI fragment, under the control of the tipA promoter, into pGM190, yielding pYT90.
After breaking the cells by using a French press, the subcellular membrane fraction was isolated as described previously (Reuther et al., 2006a). Integral membrane proteins were solubilized in Triton X-100 extraction buffer (25 mM Tris/HCl, pH 7.5, 20 % (w/v) glycerol, 1 M NaCl, 2 % Triton X-100). Unsolubilized membrane debris was removed by centrifugation (100 000 g, 30 min), and the supernatant containing the solubilized membrane proteins was loaded to a gravity flow Ni-NTA Superflow column (IBA). SpdB2-His was dialysed against 50 mM Tris/HCl, 100 mM NaCl and 1 % Triton X-100, and concentrated using Amicon Ultra-10 PL centrifugal filter devices (Millipore).
Expression and detection of SpdB3, Spd79 and SpdB2.
spdB3 and spd79 were amplified with primers SpdB3up and Spd79-flaglow, and cloned into pYT via BamHI and HindIII sites, which had been incorporated into the primer sequences. Primer Spd79-flaglow also encoded a flag-tag. The resulting pYT3 plasmid encoded SpdB3 with an N-terminal Strep-tag II (Voss & Skerra, 1997), and Spd79 with a C-terminal flag-tag (Brizzard et al., 1994). Subsequently, a 1.4 kb aac(3)IV cassette was inserted into the singular HindIII site, and the whole fragment, including the rhamnose-inducible Prham promoter, was amplified using primers Rhamup and Apralow. The PCR fragment was digested with AgeI and MscI (sites included in primer sequences), and cloned into pBG1, which had been cut with Kpn21 and MscI, generating pYT5.
Cross-linking of SpdB2.
Approximately 5 µg SpdB2-His was incubated in a total volume of 25 µl, with 0.01, 0.1 and 0.3 % glutaraldehyde, in the presence and absence of 1.25 mM DTT, for 1 h on ice. The reaction was stopped by adding 2.5 µl 1 M Tris/HCl, pH 8.0. Cross-linking was analysed by using a 10 % SDS gel and immunoblotting with Anti-His antibodies (Novagen).
Pulldown assay with StrepII-TraB.
The 1.4 kb aac(3)IV cassette was inserted into the HindIII site of pJR201 (Reuther et al., 2006a), and a 3.5 kb fragment was amplified using primers Rhamup and Apralow. Following digestion with AgeI and MscI, the respective fragment was cloned into pGB1 that had been cut with Kpn21 and MscI (Table 1), resulting in pYT7. A culture of E. coli BL21 (pYT7) was induced with 0.2 % rhamnose, harvested by centrifugation, resuspended in 1 M NaCl, 2 % Triton X-100, 25 mM Tris/HCl, pH 7.5, and 20 % glycerol, and broken by French press treatment. The strepII-TraB fusion protein was purified from the cleared lysate using StrepTactin-Sepharose (IBA), according to the manufacturer's instructions. Protein samples were analysed for copurification of SpdB2-His by SDS-PAGE and immunoblotting using anti-His (Novagen) and anti-strep-tagII antibodies (IBA).
Agarose gel shift assay.
A 0.5–1 µg quantity of DNA [pEB211, pUC18, phage 
HindIII fragments (Fermentas) and single-stranded M13 DNA] was mixed with different amounts (2.5–15 pmol) of SpdB2-His protein and reaction buffer (100 mM Tris/HCl, pH 8, 200 mM NaCl, 5 mM β-mercaptoethanol). After incubation at 24 °C for 15 min, gel loading solution (10 mM Tris/HCl, pH 7.6, 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol and 60 mM EDTA) was added, and the mixture was analysed on a 1 % agarose gel. Following electrophoresis, DNA bands were visualized by ethidium-bromide staining. As a negative control, thioesterase The1, which is involved in phosphinothricin tripeptide biosynthesis of Streptomyces viridochromogenes, was expressed with a His-tag in E. coli, and purified by Ni-NTA chromatography (S. Eys, W. Wohlleben & E. Schinko, personal communication). The purified protein, which contained additional contaminating E. coli proteins, did not show any retardation of pEB211-DNA (data not shown).
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RESULTS |
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). SpdB2, encoded by the spdB3–spd79–spdB2 operon, is the only Spd protein that has a clear homologue in all conjugative Streptomyces plasmids (Table 3). Four transmembrane helices and putative coiled-coil structures are predicted for the SpdB2 protein (Fig. 1a). spdB2 was fused with a C-terminal His6-tag-encoding sequence, and expressed in E. coli under the control of the Prham promoter (Wilms et al., 2001). Whereas SpdB2-His was synthesized as insoluble inclusion bodies at 37 °C, it was incorporated into the membrane of E. coli at an incubation temperature of 30 °C (data not shown). Minor amounts of SpdB2-His were also detected in the membrane-associated fraction. Two hours after induction of SpdB2 expression, the cultures began to lyse, demonstrating that expression of the integral membrane protein SpdB2-His was highly toxic to E. coli (data not shown).
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). Again, viability was affected after induction of spdB2-His expression. Phase-contrast microscopy revealed lysed hyphae (Fig. 2), suggesting that the incorporation of SpdB2-His into the membrane interfered with growth.
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). From 400 ml S. lividans culture, approximately 100 µg soluble SpdB2 protein was obtained.
DNA-binding activity
Since SpdB2 is involved in intramycelial DNA translocation, the capability of SpdB2-His to interact with pSVH1 DNA was studied. Various amounts of SpdB2-His (0.5–3 µg) were incubated with 1 µg pEB211 DNA, and analysed for DNA-binding activity on a 1 % agarose gel. In the presence of >1.5 µg SpdB2-His, migration of pEB211 DNA was retarded (Fig. 3a). The protein–DNA complex was too large to enter the gel, suggesting that multimers of SpdB2-His bound to the DNA. In contrast, His-tagged thioesterase The1, used as a negative control, did not show any DNA-binding activity (data not shown). The DNA-binding activity of SpdB2-His was not specific for plasmid pSVH1, since nonspecific DNAs, such as pUC18 plasmid DNA or phage DNA, were also shifted (data not shown). Interestingly, SpdB2-His did not bind to single-stranded M13 phage DNA (Fig. 3b).
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), where SpdB2-His was localized (Fig. 1b). This suggests that Spd79-flag interacts in vivo with the integral membrane protein SpdB2-His.
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). To analyse whether TraB interacts with the integral membrane protein SpdB2, probably forming a multimeric protein complex at the septal cross-walls, a strepII-TraB fusion protein was coexpressed with SpdB2-His in E. coli (pYT7). After induction of gene expression, the membrane proteins were solubilized with 2 % Triton X-100 and 1 M NaCl, and purified by StrepTactin affinity chromatography. The washing and the elution fractions were analysed by SDS-PAGE and immunoblotting for the presence of StrepII-TraB and SpdB2-His. Elution fractions that contained StrepII-TraB also contained SpdB2-His (Fig. 6). In contrast, when crude extract containing only SpdB2-His was applied to StrepTactin-Sepharose, SpdB2-His was not detected in the elution fractions (data not shown). The copurification of SpdB2-His with StrepII-TraB demonstrated a tight interaction of SpdB2 and TraB, indicating that TraB is also involved in intramycelial plasmid spreading.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This is believed to be the first report on the characterization of Spd Proteins that are required for intramycelial plasmid translocation.
Plasmid spreading on agar plates is manifested by growth retardation zones, called pock structures, which indicate the transconjugant areas (Bibb et al., 1981; Kieser et al., 1982). The size of a pock structure suggests that starting from a single germinating spore, several hundred transfer or spreading events have to take place during one round of conjugation. These inhibition zones are the result of unregulated expression of the tra and spd genes (Grohmann et al., 2003). TraB of many Streptomyces plasmids represents a kill function (Kataoka et al., 1991; Hagège et al., 1993). Also, expression of genes involved in pock formation can be detrimental. KilB of plasmid pIJ101 has been characterized as a kill-function protein (Kendall & Cohen, 1987; Pettis et al., 2001), and in this study we showed that inducing expression of the pSVH1 spdB2 gene influenced viability of E. coli and S. lividans. Although the rationale for the observed toxicity of the TraB and SpdB proteins is unclear, it can be speculated that overexpression of a putative pore-forming membrane protein may result in cell lysis.
Several plasmid-encoded Spd proteins are involved in intramycelial plasmid spreading. Interestingly, these proteins are highly diverse in different plasmids. Some of them are very small (50–90 aa), and do not show any sequence similarity to Spd proteins encoded by other plasmids (Grohmann et al., 2003).
Although the SpdB2 homologues of different Streptomyces plasmids do not possess conserved sequence motifs, they are encoded by nearly all Streptomyces plasmids (Grohmann et al., 2003). Surprisingly, even the actinophage PhiC-31 contains a SpdB2 homologue, which might be involved in the intramycelial spreading of phage DNA during infection (Smith et al., 1999). Despite the lack of sequence similarity, the SpdB2 homologues show conserved features (Table 3), with most homologues containing four transmembrane helices. As expected for an integral membrane protein, SpdB2-His of plasmid pSVH1 was detected in the membrane fraction of E. coli, and could not be solubilized by 1 M NaCl, but it was solubilized by 2 % Triton X-100, demonstrating a tight interaction with the membrane. Many SpdB2 homologues possess coiled-coil structures, and contain imperfect 4–5 aa repetitive sequences. Such structures are often involved in protein–protein interaction (Fong et al., 2004). Cross-linking experiments with SpdB2-His showed oligomerization. Bands corresponding to dimers, tetramers and higher molecular mass oligomers were detected. For seven of 13 SpdB2 proteins, a signal peptide was predicted. For SpdB2sl from pSLS, and SpdB2 of pJV1, a Pfam TolA domain was identified (http://smart.embl-heidelberg.de/). TolA is involved in the uptake of colicins and single-stranded phage DNA (Click & Webster, 1998).
Genetic organization of the spd genes in operons with translationally coupled genes suggests a cooperating function of the respective proteins. Indeed, we showed that the presence of the integral membrane protein SpdB2 directed the soluble Spd79 protein to the membrane, suggesting that SpdB2 is required for correct localization of Spd79. Furthermore, SpdB2 seemed to stabilize Spd79, because higher concentrations of Spd79 were detected when spdB2 was co-expressed.
Since the Spd proteins are involved in DNA translocation via the septal cross-walls, interaction of one of the Spd proteins with DNA was assumed. Such DNA-binding activity was demonstrated for the integral membrane protein SpdB2. In these experiments, SpdB2-His of pSVH1 did not specifically interact with a specific pSVH1 sequence, as was the case for TraB of pSVH1 that recognized the clt locus, which is required for conjugal pSVH1 transfer (Reuther et al., 2006a). SpdB2 bound to any double-stranded DNA, irrespective of its conformation or origin. At the moment, we cannot fully exclude that a specific binding activity of SpdB2-His was lost during membrane extraction and purification of the protein. Interestingly, SpdB2 bound dsDNA only, and did not interact with single-stranded M13 DNA; this supports the concept of dsDNA transfer during Streptomyces conjugation (Reuther et al., 2006a; Possoz et al., 2001).
From sequence analysis, none of the Spd proteins is predicted to have enzymic activity. Since DNA translocation to neighbouring mycelial compartments is a transport process requiring energy, the involvement of a motor protein has to be postulated. The only plasmid-encoded molecular motor protein is the septal DNA translocator ATPase TraB, which promotes plasmid transfer at the hyphal tip (Reuther et al., 2006a). As we showed in a pulldown assay, TraB interacted with SpdB2. This suggests that TraB not only mediates the primary plasmid transfer from the donor to the recipient, but also has a major role during intramycelial plasmid spreading. A TraBpSG5-EGFP fusion protein has been shown to be localized to the hyphal tips (Reuther et al., 2006a), indicating that conjugation in Streptomyces takes place at the tips of the mycelium. In contrast, the plasmids have to be translocated via the septal cross-walls during plasmid spreading. This implies that the TraB protein, normally localized at the tip, has to be redirected to the septal cross-walls. An interesting question is whether the proposed redirection of TraB can be visualized in an experimental approach by coexpression of SpdB2pSG5 with TraBpSG5-EGFP.
We propose in a speculative model that plasmid spreading is mediated by a multiprotein complex at the septal cross-walls. Oligomers of SpdB2, together with Spd79 and probably other Spd proteins, form a membrane-traversing channel. TraB interacts with this complex, and pumps dsDNA through the Spd channel to the neighbouring mycelial compartment.
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ACKNOWLEDGEMENTS |
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Edited by: J. Anné
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REFERENCES |
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Received 20 December 2006;
revised 3 May 2007;
accepted 3 May 2007.
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