Unique conjugation mechanism in mycelial streptomycetes: a DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip

A single plasmid-encoded protein, the septal DNA translocator TraB, is sufficient to promote conjugal plasmid transfer in mycelial streptomycetes. To analyse the molecular mechanism of conjugation the closely related TraB proteins from plasmids pSG5 of Streptomyces ghanaensis and pSVH1 of Streptomyces venezuelae were characterized. TraB of pSG5 was expressed as a fusion protein with eGFP and found to be localized at the hyphal tips of Streptomyces lividans by fluorescence microscopy, which strongly indicates that conjugation takes place at the tips of the mating mycelium. The TraB protein of pSVH1 was heterologously expressed in S. lividans with an N-terminal strep-tagII and purified as a soluble protein to near homogeneity. The purified protein was shown to hydrolyse ATP and to bind to a 50 bp non-coding pSVH1 sequence containing a 14 bp direct repeat. The protein-DNA complex was too large to enter an agarose gel, indicating that multimers of TraB were bound to the DNA. Denaturation of the protein-DNA complex released unprocessed plasmid DNA demonstrating that the TraB protein does not possess nicking activity. Our experimental data provide evidence that conjugal DNA transfer in streptomycetes is mediated by the septal DNA translocator TraB, an plasmid-encoded ATPase that interacts non-covalently with DNA and translocates an unprocessed double-stranded DNA molecule at the hyphal tip into the recipient.     

The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.     

Evidence that F-plasmid proteins TraV, TraK and TraB assemble into an envelope-spanning structure in Escherichia coli

We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra+ cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain suggested by yeast two-hybrid analysis. We propose that TraV is the outer membrane anchor for a trans-envelope, Tra protein structure required for the assembly of F-pili and possibly for other events of conjugal DNA transfer.     

The TraK accessory factor activates substrate transfer through the pKM101 type IV secretion system independently of its role in relaxosome assembly

A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ–TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the Tra_pKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.     

Analysis of the transfer region of the Streptomyces plasmid SCP2*

plJ903, a bifunctional derivative of the 31.4 kb low-copy number, conjugative Streptomyces plasmid SCP², was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.     

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.     

Identification and functional analysis of the transfer region of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6.2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames. This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids. All Tra proteins of pMEA300 thus may represent unfamiliar types. A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300. Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes. At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically. Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed.     

The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer

pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single-stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA. This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.     

A cloned ompR-like gene of Streptomyces lividans 66 suppresses defective melC1, a putative copper-transfer gene

Expression of tyrosinase in Streptomyces requires functional MelC1 protein, which is postulated to transfer copper to apotyrosinase. We have previously isolated a mutant of Streptomyces lividans, HT32, that phenotypically suppressed mutations in cloned melC1 (H.-C. Tseng and C. W. Chen, in preparation). Plasmid pLUS132, containing an ATG to ATA transition at the initiation codon of melC1, was used for cloning the suppressor gene from HT32. A 1687 bp suppressor DNA was isolated that contained two characteristic Streptomyces coding sequences: a 217-amino-acid open reading frame (cutR) and a truncated open reading frame (cutS) downstream. Subcloning analysis attributed the phenotypic suppression activity to the putative cutR gene from HT32. The putative CutR exhibited similarity to the response regulator OmpR of the osmoregulatory signal-transduction system in Escherichia coli. The truncated CutS resembled, to a lesser degree, the N-terminus of EnvZ, the histidine protein kinase counterpart of OmpR. DNA hybridizing to the cloned cutR-cutS sequence was detected in 16 other Streptomyces species. We postulate that the putative cutR-cutS operon regulates copper metabolism in Streptomyces.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation

Hybrids of the Streptomyces coelicolor conjugative plasmid SCP2* and the Mycobacterium plasmid pAL5000 were transferred from Streptomyces coelicolor or Streptomyces lividans to Mycobacterium smegmatis mc2155 in plate crosses. Inactivation of the SCP2* transfer function did not prevent or reduce plasmid transfer. This transfer was DNase I sensitive and thus involved release of DNA from Streptomyces, followed by transformation of M. smegmatis. M. smegmatis growing on specific solid media was also transformed by pure CCC and linear plasmid DNA. Small plasmids were taken up intact but large plasmids suffered deletions. Competence developed within 24 h of incubation at 30 degrees C or 37 degrees C, and up to 400 transformants were obtained per microg of CCC plasmid DNA. Transformation frequencies were higher when M. smegmatis was co-cultivated with plasmid-free Streptomyces, but unaffected by resident homologous sequences or inactivation of recA in M. smegmatis. Spontaneous transformation was also observed with a circular Streptomyces transposable element which inserted into chromosomal sites. Transformative plasmid transfer was also shown to occur between M. smegmatis strains. This is the first report of non-artificially induced, spontaneous plasmid transformation in Mycobacterium.     

Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.     

Analysis of recombination occurring at SLP1 att sites.

SLP1int is a conjugative Streptomyces coelicolor genetic element that can transfer to Streptomyces lividans and integrate site specifically into the genome of the new bacterial host. Recombination of SLP1 previously has been shown to occur within nearly identical 112-base-pair att sequences on the plasmid and host chromosome. We report here that both integrative recombination and intermolecular transfer of SLP1int require no more than a 48-base-pair segment of the att sequence and that SLP1 transfer occurs by a conservative rather than a replicative mechanism. The functions responsible for the excision of the element as a discrete DNA segment are induced during the conjugal transfer of SLP1.     

变铅青链霉菌内源线性质粒接合转移必需功能区的鉴定

通常细菌间环型质粒在接合转移过程中,单链质粒DNA在质粒内部“oriT”接合转移起始位点发生缺刻.随后,打开的单链质粒DNA通过细胞膜的Ⅳ型分泌系统转移到受体菌中.但是,链霉菌中的接合型线型质粒带有游离3′端,5′端与末端蛋白结合,因而不能以细胞-细胞间方式转移单链缺刻DNA.报道了变铅青链霉菌线型质粒SLP2衍生的环型质粒,与SLP2一样可以高频高效接合转移,并鉴定了接合转移功能区.质粒有效的接合转移功能区包含6个共转录的基因,分别编码一个Tra样的DNA转移酶、胞壁水解酶、2个膜蛋白(可以与ATP结合蛋白相互作用)和一个功能未知的蛋白质.从SalⅠR-/M-向SalⅠR/M宿主转移的质粒频率下降表明,线型和环型的质粒都是以双链的形式转移的.上述研究结果表明SLP2衍生的线型质粒和环型质粒以相似的与细胞膜/胞壁功能相关的机理进行接合转移.     

Directed transfer of large DNA fragments between Streptomyces species

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire approximately 22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.     

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.     

委内瑞拉链霉菌秦岭变种属间接合转移系统的构建及透明颤菌血红蛋白的表达

【目的】构建委内瑞拉链霉菌秦岭变种属间接合转移系统及透明颤菌血红蛋白的表达。【方法】以链霉菌广泛使用的整合型质粒pSET152和复制型pHZ1358为出发质粒,通过供体大肠杆菌(Escherichia coli)ET12567(pUZ8002)进行属间接合转移委内瑞拉链霉菌秦岭变种。【结果】确定了该变种的最佳接合转移条件;通过SOE-PCR(Splicing by overlap extension PCR)技术构建含PermE和vhb结构基因融合片段的整合型表达载体pJD100,转化ET12567(pUZ8002)后属间接合转移委内瑞拉链霉菌秦岭变种。通过PCR和CO结合差光谱验证了vhb基因在委内瑞拉链霉菌秦岭变种中的整合表达。【结论】本文首次探索了委内瑞拉链霉菌秦岭变种接合转移系统,确定了委内瑞拉链霉菌秦岭变种的最佳接合转移条件,并采用基因工程手段使vhb基因在委内瑞拉链霉菌秦岭变种中获得表达。     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

不吸水链霉菌梧州新亚种限制-修饰系统初探

采用菌丝体原位包埋方法和高压脉冲电泳技术从不吸水链霉菌梧州新亚种(Streptomyces ahygroscopicus wuzhouensis neosubsp. 11371)中分离得到两条质粒DNA带.通过双向电泳证明,2个质粒均为线性分子,按照分子量大小依次命名为pSAL1、pSAL2.并对不吸水链霉菌梧州新亚种的限制-修饰系统进行初步探讨:将来自变铅青链霉菌TK54的高拷贝质粒pIJ702转化不吸水链霉菌梧州新亚种原生质体,未能得到转化子,改用pIJ702转化不吸水链霉菌梧州新亚种U-3原生质体,得到了转化子.