Conjugative DNA-transfer in Streptomyces, a mycelial organism

YPLAS-02307; No of Pages 9 Plasmid xxx (2016) xxx–xxx

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Conjugative DNA-transfer in Streptomyces, a mycelial organism L. Thoma, G. Muth ⁎ Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie/Biotechnologie, Eberhard Karls Universitaet Tuebingen, Auf der Morgenstelle 28, 72076 Tuebingen, Germany

a r t i c l e

i n f o

Article history: Received 26 July 2016 Received in revised form 13 September 2016 Accepted 25 September 2016 Available online xxxx Keywords: T4SS Streptomyces Conjugation Plasmid transfer FtsK

a b s t r a c t Conjugative DNA-transfer in the Gram-positive mycelial soil bacterium Streptomyces, well known for the production of numerous antibiotics, is a unique process involving the transfer of a double-stranded DNA molecule. Apparently it does not depend on a type IV secretion system but resembles the segregation of chromosomes during bacterial cell division. A single plasmid-encoded protein, TraB, directs the transfer from the plasmid-carrying donor to the recipient. TraB is a FtsK-like DNA-translocase, which recognizes a specific plasmid sequence, clt, via interaction with specific 8-bp repeats. Chromosomal markers are mobilized by the recognition of clt-like sequences randomly distributed all over the Streptomyces chromosomes. Fluorescence microcopy with conjugative reporter plasmids and differentially labelled recipient strains revealed conjugative plasmid transfer at the lateral walls of the hyphae, when getting in contact. Subsequently, the newly transferred plasmids cross septal cross walls, which occur at irregular distances in the mycelium and invade the neighboring compartments, thus efficiently colonizing the recipient mycelium. This intramycelial plasmid spreading requires the DNA-translocase TraB and a complex of several Spd proteins. Inactivation of a single spd gene interferes with intramycelial plasmid spreading. The molecular function of the Spd proteins is widely unknown. Spd proteins of different plasmids are highly diverse, none showing sequence similarity to a functionally characterized protein. The integral membrane protein SpdB2 binds DNA, peptidoglycan and forms membrane pores in vivo and in vitro. Intramycelial plasmid spreading is an adaptation to the mycelial growth characteristics of Streptomyces and ensures the rapid dissemination of the plasmid within the recipient colony before the onset of sporulation. © 2016 Published by Elsevier Inc.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conjugative transfer of double-stranded DNA at the lateral walls . . . . . . . DNA-transfer by the FtsK-like TraB translocase . . . . . . . . . . . . . . . 3.1. Hexameric structure . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Pore forming activity . . . . . . . . . . . . . . . . . . . . . . . . 3.3. clt recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Mobilization of chromosomal markers . . . . . . . . . . . . . . . . 3.5. DNA translocation . . . . . . . . . . . . . . . . . . . . . . . . . 4. Concept of intramycelial plasmid spreading . . . . . . . . . . . . . . . . . 4.1. Detection of intramycelial plasmid spreading by fluorescence microscopy 4.2. Genes involved in intramycelial plasmid spreading . . . . . . . . . . 4.3. Evidence for a multiprotein DNA-translocation apparatus . . . . . . . 4.4. Role of TraB in intramycelial plasmid spreading . . . . . . . . . . . . 5. Transfer of linear plasmids. . . . . . . . . . . . . . . . . . . . . . . . . 6. Involvement of T4SS in the transfer of Streptomyces plasmids? . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. E-mail address: [email protected] (G. Muth).

http://dx.doi.org/10.1016/j.plasmid.2016.09.004 0147-619X/© 2016 Published by Elsevier Inc.

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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1. Introduction Streptomycetes are Gram-positive bacteria that have a key role in soil ecology due to the production of numerous hydrolytic exoenzymes (Chater et al., 2010). They have linear chromosomes with long terminal inverted repeats that are capped by terminal proteins covalently linked to the 5′-ends (Chen, 1996). With a G + C content of about 72% and a size of 8 Mb to over 10 Mb, the Streptomyces chromosomes belong to the largest bacterial chromosomes (Kirby and Chen, 2011). In contrast to most bacteria that divide by binary fission, Streptomyces developed a mycelial mode of growth by apical tip extension, which resembles growth of eukaryotic filamentous fungi (Flärdh, 2010). Peptidoglycan incorporation at the tips and new branches, which emerge in a distance of 10–40 μm to the tip, is directed by the polarisome composed of several cytoskeletal proteins (Fuchino et al., 2013). As the mycelium develops, vegetative septal cross walls form at irregular distances, often close to branching points (Flärdh, 2003). The mycelial compartments contain multiple copies of the chromosome (Ruban-Osmialowska et al., 2006). As growth is confined to the tips, chromosome replication is stimulated in the apical compartments (Ruban-Osmialowska et al., 2006). Strikingly, replisomes were shown to move at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip (Wolanski et al., 2011). Vegetative growth as multiply branching mycelium that penetrates the substrate represents the early part of the complex life cycle. Upon partial nutrient limitation, Streptomyces differentiates by producing unbranched aerial hyphae that usually grow up into the air. Only a few Streptomyces species, e.g. S. griseus or S. venezuelae, are able to differentiate also in liquid culture (Schlimpert et al., 2016). During differentiation, more or less simultaneously dozens of septa are formed in a coordinated manner, transforming the aerial hyphae into a chain of spore compartments. Following segregation of the chromosomes and thickening of the spore wall, mature spores containing a single chromosome are released giving rise to a new life cycle (Flärdh and Buttner, 2009). Conjugation in Streptomyces was discovered about 60 years ago, when Spada and Sermonti demonstrated genetic recombination in mixtures of auxotrophic mutants (Sermonti and Spada-Sermonti, 1955). Shortly after, genetic linkage of the markers (Hopwood, 1959) and involvement of plasmids (Vivian, 1971) was demonstrated. To date, a multitude of conjugative elements have been described in Streptomyces and related actinomycetes. These include middle-sized (SLP2) and very large linear plasmids (SCP1) (Chen et al., 1993; Kinashi et al., 1987), Actinomycetes Integrative Conjugative Elements (AICE) (Bibb et al., 1981; Pernodet et al., 1984; te Poele et al., 2008), low copy number plasmids, replicating via a theta mechanism, like SCP2 (Bibb and Hopwood, 1981; Haug et al., 2003) and multi-copy plasmids, using the rolling-circle replication (RCR) mode, like pIJ101, pSN22, pJV1, pSG5 and pSVH1 (Kataoka et al., 1991; Kieser et al., 1982; Muth et al., 1988; Reuther et al., 2006b; Servín-González et al., 1995). Moreover, few plasmid phages have been isolated. These elements have the genetic repertoire of a conjugative plasmid. But they also encode typical phage proteins and can be packaged into infectious phage particles (Chen et al., 2012). Chromosomes of Streptomyces and related actinomycetes are a very large reservoir of antibiotic resistance genes (D'Costa et al., 2006). As the most relevant producer of antibiotics, with the genetic potential of a single strain to produce 10–20 different metabolites (Weber et al., 2015), resistance genes probably co-evolved as part of the biosynthetic gene cluster to protect the producer from the toxic action of its own antibiotic. Therefore, the majority of all biosynthetic gene clusters also contain resistance genes whose homologues are found on resistance plasmids in pathogenic bacteria. E.g. S. griseus encodes a streptomycin-phosphotransferase in the streptomycin gene cluster, S. clavuligerus encodes a beta-lactamase in the cephamycin cluster, Saccharopolyspora erythrea has a 23S ribosomal RNA methyltransferase (Bibb et al., 1994), conferring resistance by target modification and some glycopeptide producers

contain vanHAX genes even in a similar gene organization, as also found on conjugative transposons from enterococci (Donadio et al., 2005). Beside the biosynthetic gene cluster associated resistance determinants, dozens of (putative) resistance genes are found at other sites in the chromosome. The comprehensive antibiotic resistance gene database CARD (https://card.mcmaster.ca/analyze/rgi) lists more than 50 antibiotic resistance genes for the model streptomycete S. coelicolor A3(2). The large number of antibiotic resistance genes probably reflects the need to protect the soil-dwelling streptomycetes from the numerous antibiotics produced by their competitors in the soil. With few exceptions, mainly large linear plasmids, which encode complete antibiotic biosynthetic pathways and which might frequently exchange genes with the chromosomes, Streptomyces plasmids usually do not encode antibiotic resistance genes (Grohmann et al., 2003; Vogelmann et al., 2011b). This suggests that Streptomyces plasmids might not have a selective advantage, when having picked up resistance determinants. Therefore, horizontal transfer of resistance genes in streptomycetes might not depend on the capture of resistance genes by the conjugative plasmid (see below), as it is normally the case in other bacteria.

2. Conjugative transfer of double-stranded DNA at the lateral walls Under laboratory conditions, conjugative DNA-transfer in Streptomyces takes place only on solid agar in the early growth phase, when vegetative substrate mycelium is formed. The multiply branched mycelia of donor and recipient seem to meet accidentally, since pili, aggregating proteins or other types of mating pair formation system have not been found on Streptomyces plasmids. Mating experiments with a recipient expressing the SalI restriction/modification system showed that plasmid transfer is sensitive to the presence of SalI (Possoz et al., 2001). Since SalI recognizes only double-stranded DNA, but not single-stranded DNA, the transferred DNA must be double-stranded. This contrasts the textbook knowledge, that bacterial conjugation always involves the transfer of a single-stranded DNA molecule. Plasmid transfer in Streptomyces depends on a single plasmidencoded protein, the DNA-translocase TraB (Kieser et al., 1982; Kosono et al., 1996; Servín-González et al., 1995), although it has not been ruled out yet, whether chromosomally encoded proteins, e.g. peptidoglycan (PG)-hydrolases, topoisomerases, and transporters are also involved in the transfer. Previous work showed that a TraBpSG5-fusion protein preferentially localized to the hyphal tips and probably future branching sites, which led to the assumption that conjugative plasmid transfer in mycelial streptomycetes involves the hyphal tips of the mating partners (Reuther et al., 2006a). To visualize conjugative DNA-transfer, a reporter plasmid was constructed by inserting the eGFP gene in plasmid pIJ303 (pLT303) (Thoma et al., 2016). Due to the presence of the eGFP-encoding conjugative plasmid donors showed a uniform green fluorescence, when excited with UV-light. A differentially labelled recipient showing red fluorescence was constructed by inserting mCherry into the chromosome of S. lividans. Observation of mating experiments under the microscope now allowed discrimination between plasmid carrying donor and recipient mycelium. Moreover, conjugative plasmid transfer could be observed, since transconjugants showed fluorescence in the green and in the red channel, appearing yellow in the overlay (Fig. 1). Surprisingly, in all visualized matings, donor and recipient were not in contact at the hyphal tips, but at the lateral walls (Thoma et al., 2016). This suggests that conjugative plasmid transfer in Streptomyces takes place at the lateral wall, possibly at newly developing branching sites. A closer inspection of the contact site never detected mCherry fluorescence on the donor site. This implies that during conjugation donor and recipient mycelia do not completely fuse, which would lead to a cytoplasmic flow between the fused compartments (Thoma et al., 2016). Apparently, conjugative plasmid transfer depends on a TraB membrane

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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Fig. 1. Visualization of conjugative transfer by fluorescence microscopy. To visualize plasmid transfer the reporter plasmid pLT303 was constructed by inserting eGFP into the conjugative pIJ303 plasmid (Thoma et al., 2016). Spores of the donor S. lividans TK23 (pLT303) (green) and the recipient S. lividans T7–mCherry (red) were mixed and plated. After 39 h of growth at 29 °C cells were imaged by fluorescence microscopy (A). Transconjugant hyphae appear yellow in the overlay of the red/green channel. Plasmid transfer was observed when donor and recipient were in contact at the lateral wall (B). Bar = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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plasmid transfer involves the DNA-transfer between two distinct mycelia. This implies that the plasmid-DNA has to be translocated across the cell envelopes of donor and recipient. The TraB proteins of different Streptomyces plasmids are an extremely diverse family of proteins, in part showing less than 20% sequence similarity. Nevertheless, with very few exceptions, all TraB proteins have identical domain architectures with an N-terminal membrane anchoring domain, containing one or two transmembrane helices which is followed by the central DNA-translocase domain, containing the PfamFtsK_SpoIIIE motif with Walker A and B ATP-binding boxes (Vogelmann et al., 2011a). The ATP-binding sites are essential for TraB function, as it was demonstrated for TraB of pSN22 (Kosono et al., 1996). The C-termini of TraB proteins form winged helix-turn-helix folds (γ-domain), which were shown to recognize specific plasmid sequences (see below). Interestingly, this DNA-binding domain is missing in the shorter TraB proteins of many AICE, e.g. TraSA of pSAM2. Apparently, the short TraB proteins of AICEs recognize their target DNA in a different manner. Crosslinking experiments, gel filtration chromatography and electron microscopy of purified TraBpSVH1 revealed the formation of hexamers with a central pore structure (Vogelmann et al., 2011a), as it was also described for FtsK. Consistent with the EM images, homology modeling of the TraBpSVH1 DNA translocase domain using the 3D-structure of P. aeruginosa FtsK (Massey et al., 2006) as a template revealed a 3 nm diameter for the central pore (Vogelmann et al., 2011a). This is sufficient to accommodate a double-stranded DNA molecule. 3.2. Pore forming activity

pore, which is specific for plasmid DNA, but does not allow diffusion of fluorescence proteins. 3. DNA-transfer by the FtsK-like TraB translocase traB genes of many Streptomyces plasmids have been originally identified as a kil function, depending on the presence of the kil-override kor function (Hagège et al., 1993; Kataoka et al., 1994; Kendall and Cohen, 1987; Stein et al., 1989). Since the kor function encodes a transcriptional repressor of the GntR family (Kataoka et al., 1994; Rigali et al., 2002), one can speculate that transcription of traB (kil) is initiated upon signaling a low molecular weight ligand released from the recipient. However, evidence for sensing of a signal has been only detected in the AICE pSAM2. pSAM2 encodes a nudix hydrolase (Pif), providing immunity to pSAM2 transfer when expressed in the recipient (Possoz et al., 2003). For autonomously replicating Streptomyces plasmids, no evidence for the control of traB expression by the presence of a recipient was obtained (Pettis and Cohen, 1996; Sepulveda and Muth, unpublished). Maybe induction of traB expression occurs only in that specific compartment, which is getting in contact with the recipient. This type of regulation would be missed by classical gene expression analyses and could be only proved by sophisticated fluorescence microscopy studies with reporter constructs. 3.1. Hexameric structure Despite the absence of clear sequence similarity, TraB resembles bacterial FtsK/SpoIIIE proteins in domain architecture, structure, enzymatic activity and mode of DNA recognition (Vogelmann et al., 2011a). Since FtsK/SpoIIIE are septal DNA-translocator proteins that segregate newly replicated chromosomes during cell division or sporulation to the daughter cells through a closing septum (Bigot et al., 2007; Graham et al., 2010; Massey et al., 2006), it was concluded that Streptomyces adapted the bacterial chromosome segregation system for plasmid transfer (Sepulveda et al., 2011). Whereas chromosome segregation involves translocation between two cellular compartments, conjugative

As described above, traB was initially identified as a kil function (Kendall and Cohen, 1987). An explanation for the toxicity of unregulated traB expression was obtained, when interaction of purified TraB with artificial membranes was studied. Single channel recordings in planar lipid bilayers with highly purified TraB protein of plasmid pSVH1 demonstrated that TraB is able to spontaneously insert into membranes and to form pore structures causing characteristic stepwise changes in the current flow (Vogelmann et al., 2011a). Pore formation of TraB offers the possibility that the DNA is threaded through the hexameric DNA translocase domain and pumped through the pore structure across the membrane. In this aspect, TraB might differ from FtsK, which is thought not to form a membrane pore (Dubarry and Barre, 2010), although this has never been conclusively addressed. 3.3. clt recognition Besides traB, conjugative plasmid transfer requires a second plasmid region, the cis-acting locus of transfer clt (Kieser et al., 1982). clts are small non-coding sequences of about 50–100 bp in size (Franco et al., 2003; Pettis and Cohen, 1994; Servin-Gonzalez, 1996). clts are bound by the corresponding TraB protein, without getting processed (Ducote and Pettis, 2006; Reuther et al., 2006a). Thus a clt is fundamentally different from an origin of transfer (oriT), which includes the nicking site for the synthesis of the transferred single-stranded DNA molecule (Zechner et al., 1997). Specificity of clt recognition is determined by 5–15 imperfectly conserved 8-bp direct repeats present in the clt regions of conjugative plasmids (Fig. 2). In vitro, TraB was also able to bind to synthetic clt sequences, containing fewer repeats, albeit with reduced affinity compared to the full length clt (Reuther et al., 2006a; Vogelmann et al., 2011a). The 8-bp sequence motifs are recognized by helix α3 of the wHTH fold of TraB (Vogelmann et al., 2011a), similar to the recognition of the 8-bp FtsK-Orienting-Polar-Sequence KOPS, by FtsK (Löwe et al., 2008; Sivanathan et al., 2006). Exchange of only 13 codons of traBpSVH1 corresponding to helix α3 against the respective sequence of traBpIJ101 was sufficient to switch specificity of the chimeric TraB protein which did no longer bind to the cltpSVH1 but specifically bound cltpIJ101 (Vogelmann et al., 2011a).

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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speculate that the chromosomal clt-like sequences clc are an adaption of Streptomyces to make use of the widespread conjugative plasmids. Since Streptomyces plasmids themselves do not encode any obvious beneficial traits, the development of clcs might be an alternative way of the host cell to distribute and acquire more efficiently potentially useful chromosomal genes. 3.5. DNA translocation

Fig. 2. Plasmid clts and clt-like sequences (clc) in actinomycetes chromosomes. The cisacting locus of transfer (clt) containing imperfectly conserved 8-bp direct repeats lies downstream of traB (blue) on many Streptomyces plasmids, as pSVH1 or pJV1 (A). cltlike sequences containing four copies of the respective repeat are frequently found on Streptomyces chromosomes, but are rare or absent from other actinomycetes chromosomes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The topology of the transferred DNA-molecule is unclear. TraB is the only plasmid-encoded protein, which is essential for conjugative DNAtransfer, but binding of TraB to the plasmid clt in vitro does not result in processing of the plasmid (Reuther et al., 2006a). Although difficult to envisage, this would suggest that TraB is able to translocate a circular double-stranded DNA molecule. This is supported by the observation of Wang and Pettis, that TraB of plasmid pIJ101 only promotes transfer of circular pIJ101 derivatives, but not those engineered to propagate in a linear conformation by the attachment of telomeric ends (Wang and Pettis, 2010). Recently a, model was proposed in which DNA-translocases like FtsK or TraB use the “revolution mechanism without rotation” for DNAtranslocation. In this model the double-stranded DNA molecule travels through the inner side of the central channel with one functional subunit of the DNA-translocase contacting the DNA at a time. ATP hydrolysis causes conformational changes of the DNA-translocase domain, which carries the DNA backbone to the next functional subunit inside the same ring by a sequential hand-off mechanism, so that only one subunit of the hexameric ring contacts the double-stranded DNA at a time (De-Donatis et al., 2014; Guo et al., 2016). 4. Concept of intramycelial plasmid spreading

3.4. Mobilization of chromosomal markers Mating experiments have been used for a long time to establish a detailed genetic map of S. coelicolor (Hopwood, 1967; Kieser et al., 1992). Streptomyces plasmids can mobilize chromosomal markers with a frequency of about 0.1% (Hopwood and Kieser, 1993). However, the chromosome mobilizing activity (cma) does not rely on a physical interaction of the conjugative plasmid with the host chromosome, the paradigm of HFR matings. With the exception of AICE that are integrated into a chromosomal attachment site, integration of a conjugative plasmid during conjugation was never observed. Moreover, clt, the plasmid sequence that is bound by the DNA-translocase TraB is only required for plasmid transfer, but was shown to be dispensable for cma (Pettis and Cohen, 1994), suggesting that Streptomyces chromosomes contain their own clt loci. Indeed, PatScan analyses (Dsouza et al., 1997) of Streptomyces chromosomes for the presence of putative TraB binding sites identified multiple clt-like chromosomal sequences (clc). For two of them, TraBpSVH1 binding was demonstrated in gel retardation experiments (Vogelmann et al., 2011a). Moreover, mating experiments demonstrated that the cloned clc fragments directed mobilization of a non-conjugative plasmid (Fuchs and Muth, unpublished). Strikingly, the majority of clcs were found as highly repetitive in frame insertions within coding regions, more or less randomly distributed over the whole genome. Since other Streptomyces species carried the same genes without the clc insertion, the insertion probably does not substantially interfere with the function of the respective protein (Sepulveda et al., 2011). Interestingly, occurrence of clcs seems to be restricted to streptomycetes and closely related actinomycetes, which possess Streptomyces-like plasmids. Other Actinobacteria, like Mycobacterium or Corynebacterium, which do not contain TraB encoding plasmids, do not have clc insertions in their chromosome (Fig. 2). The mechanism how the clcs have been acquired and the selection pressure preventing their elimination are unknown so far. However, one can

Conjugation in a mycelial organism differs considerably from that of unicellular bacteria. Since Streptomyces grows by apical tip extension, only apical compartments elongate and have a chance to find a mating partner. Older parts are less active, comparable with the stationary phase. This implies that if a plasmid has been transferred from a donor into an old mycelial compartment of the recipient, it would be confined in a quasi “inactive” region unable to get in contact with further mating partners and to propagate. Conjugative plasmids solved this dilemma by evolving the ability to spread within the recipient mycelium. Since mycelial compartments are separated from the neighboring compartments by cross walls (Jakimowicz and van Wezel, 2012), consisting of membranes and a PG layer, one has to propose that during plasmid spreading the plasmid has to cross these barriers. The process of plasmid spreading was discovered through the observation of pock structures associated with the conjugative plasmid transfer in streptomycetes (Bibb et al., 1978). When few plasmid carrying spores germinate on a lawn of plasmid-free spores, pocks of up to 3 mm in size develop (Hopwood and Kieser, 1993; Kieser et al., 1982). Pocks represent circular growth inhibition zones surrounding the original plasmid donor, where sporulation of the recipient is retarded. Pocks exactly match the transconjugant zone and indicate the recipient area, which was colonized by the plasmid. Identification of plasmid mutants that exhibited tiny pocks but transferred at wild type levels in mating experiments revealed the existence of spd genes (Kataoka et al., 1991; Kieser et al., 1982) and ruled out successive rounds of matings as the sole reason for plasmid spreading within the pock. The most plausible explanation for pock formation was then that, following the initial transfer from the donor, the transferred plasmid efficiently spreads within the recipient mycelium (Hopwood and Kieser, 1993; Kataoka et al., 1991). The molecular reason for the growth inhibition within the pock is unclear, but boosts of traB (kil) expression when entering the recipient might be involved (Kendall and Cohen, 1987). This could result in the formation of excess TraB membrane pores, which in consequence might inhibit growth.

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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4.1. Detection of intramycelial plasmid spreading by fluorescence microscopy For a long time intramycelial plasmid spreading has been a hypothetical model. Use of the GFP-encoding reporter plasmid pLT303 and a differentially labelled recipient strain in mating experiments provided the first experimental support for the model of plasmid spreading. Fluorescence microscopy revealed that pLT303 was able to spread over large distances in the recipient mycelium (Fig. 1). Plasmid pLT303 did not only spread into newly developing hyphae, but also reached older mycelial compartments, sometimes even ending up in the original spore from which the recipient mycelium developed (Thoma et al., 2016). On its route from the contact site of donor and recipient to the spore the plasmid had to pass multiple cross walls, which are usually formed at distances of 10–20 μm. In contrast, deletion of spd genes (pLT303ΔSpd) strongly impaired intramycelial plasmid spreading, consistent with the small pock phenotype of spd mutants (Kieser et al., 1982; Thoma et al., 2016). 4.2. Genes involved in intramycelial plasmid spreading Intramycelial plasmid spreading depends on the presence of spd genes. Inactivation of any spd gene, did not substantially interfere with the initial transfer from the donor into the recipient but reduced pock sizes to very small inhibition zones surrounding the donor (Kataoka et al., 1991; Kieser et al., 1982; Reuther et al., 2006b; Servín-González et al., 1995). Detailed deletion analyses of several RCR plasmids identified three spd genes on each plasmid (pSVH1: spdB3, spd79, spdB2; pIJ101: spdA, spdB, kilB; pJV1: spdB1, spdB2, spdB3) as being essential for intramycelial plasmid spreading (Thoma et al., 2015; Kieser et al., 1982; Servín-González et al., 1995). As sequence analysis shows, all conjugative Streptomyces plasmids contain (predicted) spd genes, often organized in transcriptional units with overlapping stop and start codons indicating translational coupling. With respect to the gene organization of the spd genes, the plasmids (or their transfer regions) fall into distinct families (Fig. 3), named after the best characterized representatives. In nearly all plasmids, the spd/tra operon is under the transcriptional control of the divergently transcribed GntR-type regulatory gene traR (korA). The striking feature of Spd proteins of different plasmids is their uniqueness. Only very few spd genes, like spdB2, seem to be conserved (Fig. 3) and are present on most plasmids. In contrast, many plasmids encode putative Spd proteins, which do not share any sequence similarity to Spd proteins encoded by other plasmids (Grohmann et al., 2003; Thoma and Muth, 2015). Not a single Spd protein shows similarity to a functional characterized protein or contains a Pfam domain helping to predict its putative function in intramycelial plasmid spreading. Unfortunately, only few Spd or putative Spd proteins have been characterized biochemically to elucidate their activity. SpdA of plasmid pSN22 was reported to affect the distance of plasmid spreading (Kataoka et al., 1991). spdA-like genes of most plasmids lie next to the single-stranded origin sso for lagging strand synthesis during RCR. spdA genes are always associated with a two or three times repeated 12 bp palindromic nucleotide sequence (Servín-González et al., 1995; Thoma et al., 2014), which often coincides with a SRDD/E-X6-8-SRDD/E amino acid motif within SpdA. The purified SpdA homologue SpdA2 of plasmid pIJ101 was shown to bind the palindromic sequence. However, mutagenesis of the palindromic sequence without changing the encoded protein sequence or deletion of spdA2 affected plasmid stability and did not abolish intramycelial plasmid spreading (Thoma et al., 2014). This suggests a role of spdA in plasmid replication/stability/distribution, which could also influence efficiency of spreading, rather than a specific function in intramycelial plasmid spreading. SpdB2 is characterized by the presence of four transmembrane helices. This feature identifies putative SpdB2 homologues on nearly all conjugative Streptomyces plasmids (Tiffert et al., 2007). Deletion of

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spdB2 of plasmid pSVH1 abolished spreading and pock formation. Interestingly, induction of spdB2pSVH1 expression in E. coli BL21(pLys) resulted in the formation of spheroplasts. Since spheroplasts were not observed in the absence of either spdB2pSVH1 or pLys, it was concluded that SpdB2 forms membrane pores, which enable the T7-lysozyme encoded by pLys to degrade the PG and consequently transform the rods into spheres (Thoma et al., 2015). Purified SpdB2pSVH1 was shown to oligomerize and to form pore structures in artificial membranes. SpdB2pSVH1 bound to PG-sacculi of S. lividans and interacted with double-stranded DNA without any sequence specificity (Thoma et al., 2015; Tiffert et al., 2007). All Streptomyces plasmids encode very short orfs of less than 80 amino acids in their transfer and spd region. pFP11.19c lies next to traB on plasmid pFP11 (Zhang et al., 2008). It encodes a 61 aa protein with a single predicted transmembrane helix. pFP11.19c shows significant sequence similarity to SmeA of S. coelicolor, which was shown to localize the FtsK homologue SffA to the septal cross walls (Ausmees et al., 2007). Although pFP11.19c homologues have been identified on only few other Streptomyces plasmids, like pFP1 and pSV2, it is tempting to speculate that pFP11.19c or homologues that do not show sequence similarity are required for intramycelial plasmid spreading by localizing TraB to the cross walls. 4.3. Evidence for a multiprotein DNA-translocation apparatus The gene organization of spd genes and the identical phenotypes of spd mutants in matings suggested that the Spd proteins work together during plasmid spreading. To study which of the Spd proteins cooperate, the protein-protein interaction pattern of all proteins/orfs of plasmid pSVH1 and of plasmid pIJ101 was determined with a bacterial two-hybrid system. These analyses revealed multiple interactions for nearly all tested proteins (Fig. 4). SpdB, KilB, Tra and SpdA2 of plasmid pIJ101 and SpdB3, Spd79, SpdB2, Orf108, TraB, Spd198, and Orf140 of pSVH1 showed self-interaction, suggesting that these proteins act as dimers or oligomers (Thoma et al., 2015; Thoma et al., 2016; Vollmer and Muth, unpublished). Oligomerization of Orf140, SpdB2, and TraB was confirmed by chemical crosslinking, blue native gel electrophoresis, gel filtration analyses or electron microscopy (Thoma et al., 2015; Tiffert et al., 2007; Vogelmann et al., 2011a). Both the N-terminal 130 aa of TraB, and the C-terminal part of TraB, comprising the DNA-translocase and clt recognition domain interacted with each other. Also in case of SpdB2, both, the N-terminal coiled-coil domain (amino acids 1–99) and the C-terminal domain (amino acids 206–409) showed self-interaction suggesting that both parts of the protein contribute to oligomerization (Thoma and Muth, 2015). Several Spd proteins interacted not only with other Spd proteins, but even with proteins encoded by genes that had no clear phenotype in mating experiments, like Spd198, Orf140, and SpdA (Thoma et al., 2015). This suggests that also these proteins are as accessory proteins somehow involved in conjugative DNA-transfer or couple the conjugative transfer with other cellular processes. Strikingly, the TraB proteins of both plasmids interacted with several Spd proteins (and putative accessory proteins) of the respective plasmid, suggesting that TraB is not only crucial for the initial transfer from the donor into the recipient, but also has a role in intramycelial plasmid spreading. All these observations are consistent with a model that the Spd proteins form a multi-protein DNA-translocation apparatus together with TraB to direct intramycelial plasmid spreading. 4.4. Role of TraB in intramycelial plasmid spreading Pettis identified mutations within TraBpIJ101 that specifically reduced pock size without dramatically affecting the initial transfer rate (Pettis and Cohen, 2000), indicating a role of TraB in DNA-translocation events during plasmid spreading. Moreover, none of the Spd proteins is predicted to be a motor protein, able to move DNA. However,

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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Fig. 3. Gene organization of conjugative plasmids from Streptomyces and related actinomycetes. Complete plasmids or their transfer and spread regions, respectively are drawn in scale, starting from the GntR-type regulatory gene. Identical colors indicate identical function. In case of absence of sequence similarity, function was proposed according to the predicted secondary structure or presence of characteristic protein domains. Spd genes are named according to the nomenclature of pJV1 (Servín-González et al., 1995). int/xis: integrase/ excisionase, ltg: lytic transglycosylase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

demonstration of the specific role of TraB in intramycelial plasmid spreading is complicated, since traB is essential for the conjugative transfer from the donor into the recipient, preceding intramycelial plasmid spreading. To investigate whether TraB also directs plasmid transfer within the recipient mycelium, traB was deleted from the conjugative pIJ101 derivative pIJ303 (Thoma et al., 2016). Mating experiments with a donor strain carrying a copy of traB under control of the repressor korA integrated into the chromosome and plasmid pIJ303ΔTra yielded transconjugants, since the chromosomally-encoded traB copy could complement the defect of pIJ303ΔTra and direct its transfer to the recipient. However, pIJ303ΔTra was unable to spread in the recipient and only tiny pocks were formed. When both strains, donor and

recipient, contained traB integrated into the chromosome, larger pock structures were observed, indicating that pIJ303ΔTra spread, when TraB was provided in the recipient (Thoma et al., 2016). This genetic proof confirmed the mutational analyses (Pettis and Cohen, 2000) and the protein-protein interaction data and demonstrated that intramycelial plasmid spreading depends on the presence of TraB in the recipient mycelium. 5. Transfer of linear plasmids Although linear plasmids encode typical TraB proteins, conjugative transfer of a linear plasmid might functionally differ from that of a

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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Fig. 4. Interaction network and predicted structure and localization of plasmid-encoded proteins of plasmids pIJ101 and pSVH1. All orfs were tested for protein-protein interaction using the adenylate cyclase based bacterial two-hybrid system (Karimova et al., 1998). Topology of membrane proteins was predicted with TMPred (Hofmann, 1993). Oligomerization is based on chemical crosslinking, electron microscopy or blue native PAGE (Thoma et al., 2015; Thoma et al., 2016; Vogelmann et al., 2011b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

circular molecule. The linear replicons of Streptomyces are capped by terminal proteins, which are covalently bound to the 5′-ends of the DNA (Kirby and Chen, 2011). Conjugative transfer of such molecules would require that the ~ 20 kDa terminal proteins fit into the TraB DNA-translocation pore. Alternatively, one would have to propose an enzymatic activity associated with the conjugative transfer that removes and reattaches the terminal proteins. For the linear 50-kbp plasmid SLP2 of S. lividans it was shown that inactivation of traB abolished its transfer (Hsu and Chen, 2010). In addition to traB, a helicase-like gene, ttrA, located in the subtelomere region of SLP2 and that of other linear replicons was found to be involved in transfer. Inactivation of ttrA blocked conjugative transfer, demonstrating that SLP2 transfer depends on ttrA (Huang et al., 2003). The mechanistic differences in the transfer of circular and linear plasmids are further supported by the finding that linear plasmids are only able to mobilize linear chromosomes. If the chromosome was artificially circularized by joining the ends, the linear plasmid SLP2 was unable to mobilize chromosomal markers. In contrast, circular plasmids were able to mobilize both types of chromosomes (Lee et al., 2011). 6. Involvement of T4SS in the transfer of Streptomyces plasmids? Very few plasmids of mycelial actinomycetes encode certain components of type IV secretion systems (T4SS), characteristic for conjugation in unicellular bacteria. Streptomyces plasmids pFP11 and pFP1 encode conjugative relaxases in addition to TraB and Streptomyces-type Spd proteins (Zhang et al., 2008). Such relaxase genes are also found on the Nocardiopsis plasmid pSQ10 (Zeng et al., 2011) and the Amycolatopsis plasmid pA387, which in addition encodes a VirD4-like coupling protein (Malhotra et al., 2008). But functionality of any of these relaxases in plasmid transfer has not been demonstrated. Moreover, the relaxase (and the product of the relaxase reaction) would require a functional T4SS not encoded on the respective plasmids. The 128 kb Streptomyces plasmid pZL1 was reported to contain two distinct transfer regions, both functional. A DNA fragment containing seven genes, including traB, mediated plasmid transfer in S. lividans at a frequency of 5 × 10−1, when cloned onto a non-conjugative plasmid

(Zhao et al., 2014). Surprisingly, also a second pZL1 fragment, located ~ 50 kbp away from the traB region was able to mediate conjugative plasmid transfer at a similar frequency. This fragment encoded a TraAlike conjugative relaxase, a 184 aa hypothetical protein, and a putative VirD4-like coupling protein. Other T4SS genes with similarity to virB4 or virB6 are located next to the virD4-like gene, but they were not required for the conjugative plasmid transfer. Since it is very unlikely that a conjugative relaxase and a coupling protein can transfer a plasmid by their own, one has to propose the involvement of a functional T4SS, encoded on the chromosome of S. lividans, which is able to mobilize these plasmids. A thorough analysis of the S. lividans and the related S. coelicolor chromosomes for genes encoding proteins with structural similarity to T4SS proteins identified two clusters of genes in each strain. Interestingly, one of these clusters has homologues (SLP2.19–23) on the linear plasmid SLP2 (Chen et al., 1993). This cluster (SCO4126–4132) has been already characterized in S. coelicolor and named cmdABCDEF and ttgA, respectively (Xie et al., 2009). Single mutations in either one of the cmd genes caused a defect in proper sporulation and affected production of the blue-pigmented antibiotic actinorhodin, but did not affect transfer of the SLP2 derivative pQC542 (Xie et al., 2009).

7. Conclusion Mycelial organisms like Streptomyces are non-motile and can only reach a nutrient-rich environment by extending hyphal tips. Older parts of the mycelium are confined in a less favourable environment if the available nutrients have been used up. Consequently, a conjugative plasmid entering a mycelial organism has to face the problem, how to avoid being sealed off in a dead end street, meaning in mycelial compartments that are unable to continue growth. A solution for this problem was the development of a secondary DNA-translocation system, the intramycelial plasmid spreading. This system allows the plasmid to travel within older parts of the mycelium to reach the actively growing tip compartments or differentiating hyphae. Since aerial hyphae, which become transformed into spore chains develop on the cost of the

Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004

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autolysing substrate mycelium, intramycelial plasmid spreading greatly enhances the probability of the plasmid being packaged into spores. In a mycelial organism, the plasmid can spread by three mechanistically distinct ways. First, the plasmid replicates in the tip compartment and copies of the plasmid are segregated before septal cross walls are formed. Second, intramycelial plasmid spreading translocates copies of the plasmid across a septal cross wall to the next mycelial compartment. And third, since recipient compartments which have obtained a plasmid by anyone of the two mechanisms can act as donor, if they sense a suitable recipient, they can transfer the plasmid in further rounds of conjugation. During plasmid spreading the plasmid has to travel over a distance of more than 10 μm to reach the next compartment. It is unknown, whether the plasmid passively moves by diffusion or whether active processes involving cytoskeletal elements are used under certain conditions. Orf140 of plasmid pSVH1, which was shown to interact to higher oligomeric structures (Thoma et al., 2015) could be a candidate for such a cytoskeletal element. Although the exact nature of septal cross walls has not been elucidated, imaging techniques demonstrated the presence of membranes and PG. Septal cross walls seem to represent a barrier for plasmid DNA, as shown by the inability of pLT303ΔSpd to spread within the recipient mycelium. Therefore, the most plausible role of the Tra/Spd apparatus is to promote DNA- translocation across septal cross walls. Several lines of evidence support this assumption. i. most of the involved proteins contain transmembrane helices and are associated with the membrane. ii. several of these proteins bind DNA and PG. iii. SpdB2 forms pores in vivo and in vitro. iv. SpdB2 interacts with the DNA-translocase TraB. These observations fit into the following model of the conjugative plasmid transfer in mycelial actinomycetes. Donor and recipient mycelia get accidentally into contact at the lateral wall, possibly at new branching points. At the contact site, expression of the traB gene is induced in the donor compartment. Involving chromosomallyencoded PG-hydrolases, PG of donor and recipient is locally degraded. TraB oligomerizes and forms a hexameric pore structure in the membrane. TraB might also promote fusion of donor and recipient membranes, similar to the SpoIIIE promoted membrane fusion during sporulation of B. subtilis (Sharp and Pogliano, 1999). The C-terminal wHTH domain of TraB binds to the specific 8-bp TRS repeats in the plasmid clt. Under ATP consumption the double-stranded plasmid DNA is translocated by the revolution mechanism inside the TraB channel. Whether transfer of the circular plasmid DNA requires processing of the DNA, maybe by recruitment of a chromosomally-encoded topoisomerase, is unknown. In the new cellular environment upon entering the recipient, traB and spd genes are induced and a multi-protein DNAtranslocation apparatus is inserted into the septal cross wall of the neighboring compartment. The DNA-translocase TraB then pumps the plasmid DNA through the septum traversing Spd-complex, resulting in the efficient colonization of the recipient mycelium by the incoming plasmid. If a plasmid reaches a new compartment, traB and spd genes are induced, since the TraR/KorA repressor is not yet synthesized. This boosts further plasmid-translocation events, resulting in intramycelial plasmid spreading or a further round of conjugative DNA-transfer. Despite considerable progress in elucidating the unique conjugative DNA-transfer system of mycelial growing Streptomyces (Thoma and Muth, 2015), many more issues have to be addressed to fully understand Streptomyces conjugation and its relevance in soil. A key approach is certainly the direct visualization of plasmid DNA in real time. Simultaneous localization of TraB and Spd-proteins together with the plasmid DNA, e.g. by fluorescence repressor operator systems (FROS) and high resolution time lapse microscopy of conjugating hyphae will reveal kinetics of the transfer, starting from the induction of TraB expression in the donor, the formation of a DNA-translocation pore to the recipient, replication and migration of the plasmid within the recipient mycelium ending up in the translocation of the plasmid DNA across the septal cross walls.

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Please cite this article as: Thoma, L., Muth, G., Conjugative DNA-transfer in Streptomyces, a mycelial organism, Plasmid (2016), http://dx.doi.org/ 10.1016/j.plasmid.2016.09.004