Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3^r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.     

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

Physical map of the linear chromosome of Streptomyces hygroscopicus 10-22 deduced by analysis of overlapping large chromosomal deletions

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.     

变铅青链霉菌与放线菌噬菌体φHAU3相互关系的研究

变铅青链霉菌ＺＸ１,是由变铅青链霉菌ＪＴ４６经ＮＴＧ诱变后产生的一株修饰基因突变株,与其亲本ＪＴ４６相比,ＺＸ１除了与ＤＮＡ降解有关的基因发生了突变（Ｄｎｄ－,即该突变株的ＤＮＡ在含有Ｆｅ２＋的缓冲液中电泳不受到降解,而野生型变铅青链霉菌在同样条件下则遭到降解）以外,对噬菌体　ＨＡＵ３的抗性也随之消失,这项特征主要表现在噬菌斑大小和成斑单位（效价）上的显著变化。研究结果表明,噬菌体　ＨＡＵ３以同等的频率吸附野生型变铅青链霉菌及其突变菌株ＺＸ１,从　ＨＡＵ３基因组中没有克隆到被变铅青链霉菌识别的特异性靶位点;噬菌体ＨＡＵ３的ＤＮＡ也可以转染野生型变铅青链霉菌原生质,但其释放的噬菌体粒子只能感染突变菌株ＺＸ１,而不能感染野生型变铅青链霉菌。噬菌体ＨＡＵ３在突变株ＺＸ１中的繁殖遵循一步生长曲线,单菌释放量大约为１００,而　ＨＡＵ３感染野生型变铅青链霉菌后,则检测不到噬菌体的释放。     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.     

Effect of cloned DNA fragment from Streptomyces chrysomallus No.2 on metabolism in Streptomyces strains

The effects on cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in streptomyces strains with various potencies in producing this antibiotic, their inactive mutants, and model strain of Streptomyces lividans 66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Cloning of the xylanase gene of Streptomyces lividans

The xylanase (xln) gene of Streptomyces lividans 1326 was cloned by functional complementation of the xylanase-negative and beta-1,4-glucan-glucanohydrolase-negative double mutant of S. lividans using the multicopy plasmid pIJ702. Three clones had a common 2-kb DNA fragment as determined by restriction mapping and Southern hybridization. These clones secreted a xylanase of Mr 43,000 which reacted with specific anti-xylanase antibodies and corresponded exactly to the enzyme previously isolated from the wild-type strain. The DNA fragment likely carried the full structural gene, the xln promoter and also the regulatory sequence, since the xylanase activity was inducible by xylan. Enzyme levels of up to 380 IU/ml of culture filtrate were obtained.     

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Physical map of the linear chromosome of Streptomyces griseus.

The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.     

头状链轮丝菌染色体复制起始区的克隆和对变铅青链霉菌的转化

头状链轮丝菌(Streptoverticillum caespitosus) ATCC27422是抗肿瘤药物——丝裂霉素A的产生菌,根据高GC含量革兰氏阳性菌染色体复制起始区两端基因序列的保守性,采用PCR方法从该菌中克隆了一段包含染色体复制起始区(oriC)的1.3 kb片段。序列分析发现,克隆片段与天蓝色链霉菌的oriC及邻近区域的同源性达80％以上;头状链轮丝菌oriC中含有22个DnaA-box,保守序列为TTGTCCACA。以克隆片段构建的质粒可以跨属转化变铅青链霉菌(S. lividans)ZX7,原生质体转化效率为3.2×10²个/μgDNA;质粒在S. lividans ZX7中能以低拷贝形式稳定存在;转化子的菌落和菌丝形态均正常。头状链轮丝菌oriC序列与几种链霉菌的oriC有较高的同源性,以及在变铅青链霉菌中仍具有复制起始活性等说明链轮丝菌属与链霉菌属在系统发生上关系较近;但采用最大似然法分析oriC序列构建的头状链轮丝菌与几种链霉菌的系统进化树表明,头状链轮丝菌与几种链霉菌之间的分化距离远大于链霉菌之间的分化距离。该证据支持链轮丝菌属的独立分属。