The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

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.     

The integrase of the conjugative transposon Tn916 directs strand- and sequence-specific cleavage of the origin of conjugal transfer, oriT, by the endonuclease Orf20

Orf20 of the conjugative transposon Tn916 was purified as a chimeric protein fused to maltose binding protein (MBP-Orf20). The chimeric protein possessed endonucleolytic activity, cleaving both strands of the Tn916 origin of conjugal transfer (oriT) at several distinct sites and favoring GT dinucleotides. Incubation of the oriT DNA with purified Tn916 integrase (Int) and MBP-Orf20 resulted in strand- and sequence-specific cleavage of oriT at a TGGT motif in the transferred strand. This motif lies immediately adjacent to a sequence in oriT previously shown to be protected from DNase I cleavage by Int. The endonucleolytic cleavages produced by Orf20 generated a 3' OH group that could be radiolabeled by dideoxy ATP and terminal transferase. The production of a 3' OH group distinguished these Orf20-dependent cleavage events from those catalyzed by Int at the ends of Tn916. Thus, Orf20 functions as the relaxase of Tn916, nicking oriT as the first step in conjugal DNA transfer. Remarkably for a tyrosine recombinase, Tn916 Int acts as a specificity factor in the reaction, conferring both strand and sequence specificities on the endonucleolytic cleavage activity of Orf20.     

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.     

Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.     

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.     

Organization of DNA sequences and replication origins at yeast telomeres

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.     

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) possesses a centrally located replication origin and shows significant homology to the transposon Tn4811.

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

Characterization of the terminal sequences flanking the transposon that carries the Escherichia coli enterotoxin STII gene

The Escherichia coli enterotoxin STII gene is flanked by two repeat sequences, approx. 600 bp each and 8 kb apart. This 9-kb DNA fragment has been shown to transpose as a unit and is thus considered a transposon. It is presently designated as Tn4521. In this study, the two terminal sequences of Tn4521 cloned in pPS1 were localized, isolated, and characterized. The two terminal sequences were found to be composed of IS2 sequences and were in an inverted repeat orientation. However, neither repeat contained a complete IS2. The LTR contained bp 1-722, whereas the RTR contained bp 17-536 and 969-1327, all three of the IS2 sequence.     

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.     

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.     

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.     

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.     

Location of determinants of stability to neomycin and kanamycin as well as DNA segments, responsible for amplification in Streptomyces plasmids pSU3 and pSU10

The S. rimosus amplifying sequence AUD-Sr1 encodes kanamycin and neomycin resistance, defined in the case of neomycin by aminoglycoside phosphotransferase. Its cloning on plasmid SLP1.2 makes possible the co-amplification of the obtained hybrid plasmids in S. lividans. In our study the regions responsible for resistance to aminoglycoside antibiotics and the capacity for amplification the two hybrid plasmids pSU10 and pSU3 were determined. Experiments on subcloning of the AUD-Sr1 sequence fragments on vector pIJ702 revealed localization of kanamycin and neomycin resistance determinants between PvuII(6) and BglII(7) on the AUD-Sr1 sequence fragments of 2.0 kb length. Two regions responsible for amplification of the hybrid plasmids were detected with deletion and insertion mapping. The first region is localized in the region of the plasmid SLP1.2 BamHI site and the second region is localized on the PstI(4)-PvuII(6) of the AUD-Sr1 sequence fragment of 1.1 kb length.