Cloning and analysis of the replication origin and the telomeres of the large linear plasmid pSLA2-L in Streptomyces rochei

The replication origin and both terminal segments were cloned from the large linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. The basic replicon consists of a 1.9-kb DNA fragment, which contains the genetic information required for autonomous replication in circular form. Sequence analysis revealed two ORFs, RepL1 and RepL2, with no similarity to any of the replication initiator proteins in the database. Deletion and mutational analysis showed that RepL1 is essential for replication and RepL2 has a subsidiary function. The origin of replication may be located 800 bp upstream of repL1. Sequencing of the left and right terminal segments revealed the presence of 12 palindromes. The sequence of the first 90 bp, including palindromes I–IV, shows great similarity to that of other Streptomyces linear chromosomes and plasmids. These results suggest that the internal replication origins of the linear replicons vary widely, in contrast to the high degree of conservation of their telomeres. Received: 2 December 1999 / Accepted: 12 April 2000     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Characterization of the genetic components of Streptomyces lividans linear plasmid SLP2 for replication in circular and linear modes

The nucleotide sequence of Streptomyces lividans linear plasmid SLP2 consists of 50,410 bp (C. H. Huang, C. Y. Chen, H. H. Tsai, C. Chen, Y. S. Lin, and C. W. Chen, Mol. Microbiol. 47:1563-1576, 2003). Here we report that the basic SLP2 locus for plasmid replication in circular mode resembles that of Streptomyces linear plasmids pSLA2 and SCP1 and comprises iterons(SLP2) and the adjacent rep(SLP2) gene. More efficient replication additionally required the 47-bp sequence between bp 581 and 628 upstream of the iterons. Replacement of either the iterons or the rep gene of SLP2 by the corresponding genes of pSLA2 or SCP1 still allows propagation in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that these plasmids share similar replication mechanisms. To replicate SLP2 in linear mode, additional SLP2 loci--either mtap(SLP2)/tpg(SLP2) or mtap(SLP2)/ilrA(SLP2)--were required. IlrA(SLP2) protein binds specifically to the iterons(SLP2) in vitro. Interactions were detected between these SLP2-borne replication proteins (Mtap(SLP2), Tpg(SLP2), and IlrA(SLP2)) and the telomeric replication proteins (TpgL, TapL, and TpgL) of the S. lividans chromosome, respectively, but the SLP2 proteins failed to interact. These results suggest that SLP2 recruits chromosomally encoded replication proteins for its telomere replication.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSO_A type, as well as with the conversion signal of the Escherichia coli phage X174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).     

A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10-22

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.     

高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

从猪粪堆肥中分离到一株编号为X3-3的可以在50℃高温生长的链霉菌菌株,该菌株含有一个约7kb的环型质粒pTSC2。【目的】克隆、测序和分析pTSC2,以及鉴定质粒的复制方式。【方法】利用分段克隆和引物延伸获得pTSC2的全序列,利用多序列比对寻找复制元件rep、dso和sso,利用中性转移和Southern杂交检测复制中间体。【结果】克隆和测序获得了全长为7516bp的pTSC2序列,预测编码8种蛋白,其中4种蛋白与链霉菌滚环复制的质粒pIJ101中负责复制和接合转移的蛋白非常相似。pTSC2的复制元件rep、dso和sso也与pIJ101的相似。克隆、转化变铅青链霉菌ZX7以及高温链霉菌2C证明了rep和dso为复制所必需元件。Southern杂交检测到pTSC2复制过程中积累了大量的单链DNA。【结论】高温链霉菌质粒pTSC2以滚环方式进行复制。这是首次在高温链霉菌中克隆和测序质粒,以及鉴定其复制方式。     

红球菌线型质粒pNSL1的克隆、测序和复制区的鉴定

从红球菌NS1中检测到两个线型质粒pNSL1和pNSL2。【目的】克隆、测序和分析pNSL1,并鉴定质粒的复制区。【方法】利用脉冲电泳方法从凝胶中回收大量的质粒DNA,进行鸟枪法克隆、测序和拼接,通过生物信息学分析和实验证明质粒的自主复制区。【结果】克隆、测序和拼接获得pNSL1全长为117252bp的序列,包括在红球菌中保守的1282bp端粒的序列。序列预测含有103个蛋白编码区,包括质粒的复制、分配、转移等功能基因。将pNSL1中一个与分枝杆菌质粒的复制基因同源的pNSL1.038及其上游的767bp非编码序列克隆到大肠杆菌质粒,电击转化珊瑚诺卡氏菌4.1037,获得了抗性转化子。【结论】克隆、测序了全长的线型质粒pNSL1,鉴定了质粒的复制区。     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus

A kinetic model describing the cephalosporin biosynthesis in Streptomyces clavuligerus was developed. Using previously reported kinetic data of biosynthetic enzymes, we examined the kinetics of cephalosporin production. The predicted time profile of the specific production rate during a batch culture parallels that of experimental observation. Sensitivity analysis reveals that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is the rate-limiting enzyme. The effect of amplifying ACV synthetase on the specific production rate was analyzed theoretically. Increasing ACV synthetase enhances the production rate initially until ACV synthetase enhances the production rate initially until deacetocycephalosporin C hydroxylase becomes rate-limiting. Such kinetic analysis can provide a rational basis for modifying the biosynthetic machinery of cephalosporin through gene cloning.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

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.     

Morphology and viability analysis of Streptomyces clavuligerus in industrial cultivation systems

Bioprocess and Biosystems Engineering -     

Genetic and biochemical analysis of the antibiotic biosynthetic gene clusters on the Streptomyces linear plasmid

We extensively analyzed the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two structurally unrelated polyketide antibiotics, lankacidin and lankamycin. It was found that amine oxidase LkcE oxidizes an acyclic amine to an imine, which is in turn converted to the 17-membered carbocyclic lankacidin. Heterologous expression and translational fusion experiments indicated the modular-iterative mixed polyketide biosynthesis of lankacidin. Concerning to lankamycin biosynthesis, starter unit biosynthesis and the post-PKS modification pathway were elucidated by feeding and gene inactivation experiments. It was shown that pSLA2-L contains many regulatory genes, which constitute the signaling molecule/receptor system for antibiotic production and morphological differentiation in this strain. Two signaling molecules, SRB1 and SRB2, that induce production of lankacidin and lankamycin were further isolated and their structures were elucidated. Each contains a 2,3-disubstituted butenolide skeleton, and the stereochemistry at C-1′ position is crucial for inducing activity.