[Show abstract][Hide abstract] ABSTRACT: Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA
gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.
Full-text · Article · Aug 2015 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Top-down reduction of the bacterial genome to construct desired chassis cells is important for synthetic biology. However, the current progress in the field of genome reduction is greatly hindered by indispensable life-essential genes that are interspersed throughout the chromosomal loci. Here, we described a new method designated as "MEGA (Multiple Essential Genes Assembling) deletion and replacement" that functions by assembling multiple essential genes in an E. coli-S. cerevisiae shuttle vector, removing targeted chromosomal regions containing essential and non-essential genes using a one-round deletion, and then integrating the cloned essential genes into the in situ chromosomal loci via I-SceI endonuclease cleavage. As a proof of concept, we separately generated three large deletions (80-205 kbp) in the E. coli MDS42 chromosome. We believe that the MEGA deletion and replacement method has potential to become widely used in large-scale genome reductions in other sequenced organisms in addition to E. coli.
No preview · Article · Dec 2014 · ACS Synthetic Biology
[Show abstract][Hide abstract] ABSTRACT: We identified the minimal locus of 163-kb plasmid pSV1 of S. violaceoruber for replication in S. lividans. This locus comprised a repA gene and an upstream 407-bp sequence containing two inverted repeats (IR-III and IR-IV) within an iteron, an AT-rich region and a 300-bp non-coding sequence (NCS). RepA protein bound specifically to a 94-bp sequence covering the intact IR-III and IR-IV to form multimers of DNA/protein complexes, but was unable to bind specifically to the NCS and the promoter of repA gene. Interestingly, this "bound" region also leaves eight 1-bp "unbound" spacers at 7-11-9-11-9-11-9-11-8 bp intervals. RepA protein-protein interaction could form dimers or trimers in vitro. These results suggest that a high-order complex between pSV1 RepA protein and the long inverted-repeats may be formed during initiation of plasmid replication. This article is protected by copyright. All rights reserved.
Preview · Article · Oct 2013 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: Previous reports showed that the large linear plasmid SCP1 of Streptomyces coelicolor A3(2) contains a 5.4-kb centrally-located replication locus. We report here that SCP1 actually contains three internal replication loci. Sub-cloning of the 5.4-kb sequence identified a 3.2-kb minimal locus (rep1A/repB/iteron) that determined propagation in S. lividans. The two newly identified replication genes, rep2A and rep3A, resemble the rep gene of Streptomyces circular plasmid pZL12. Transcription start points of the three replication genes were determined. The three replication loci could independently determine propagation in linear mode in S. lividans. Individual and sequential deletions of the rep1A and rep3A genes were successful. The SCP1-derived linear plasmids with deletions of the rep1A and/or rep3A genes still propagated in similar copy numbers, were inherited largely stable and transferred efficiently by conjugation in S. coelicolor. Interestingly, SCP1 can be artificially circularized to yield a 280-kb circular plasmid, C-SCP1, which contains the three replication loci. Strikingly, the copy numbers, inheritance and transfer of C-SCP1 resembled that of the linear SCP1 plasmids. Transcriptions of the rep1A, rep2A and rep3A genes in linear or artificially-circularized SCP1 were detected at all the time-points of strain growth.
[Show abstract][Hide abstract] ABSTRACT: The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters. This article is protected by copyright. All rights reserved.
Preview · Article · May 2013 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: Here we report that tgdA, a novel gene encoding a putative transglycosylase, affects both the morphological differentiation and the yield of blue-pigmented
compound actinorhodin in Streptomyces coelicolor. The tgdA null mutant displays sparse aerial hyphae and irregular spore chains frequently lacking chromosomal DNA. Elevated actinorhodin
production coincides with the overexpression of actII-orf4 in mutant. tgdA expression is temporally and developmentally regulated. The tgdA orthologs in Streptomyces avermilitis and Streptomyces lividans also affect differentiation.
Preview · Article · Feb 2013 · Acta Biochimica et Biophysica Sinica
[Show abstract][Hide abstract] ABSTRACT: Background
Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described.
We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex.
This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.
[Show abstract][Hide abstract] ABSTRACT: Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4.
Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used.
The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (phiCQ4) was observed by transmission electron microscopy. LinearphiCQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4.
Streptomyces plasmid pCQ4 could be transformed into lytic phagephiCQ4, and the phage segment on pCQ4 might be a mobile unit.
[Show abstract][Hide abstract] ABSTRACT: Streptomyces sp. X335, harboring a 4. 3-kb plasmid pDYM4.3k, was isolated from Tibet sample. Cloning, sequencing, analyzing and functional investigations of pDYM4. 3k.
Prime walking to obtain plasmid sequence, BLAST to predict gene function, Southern hybridization to determine replication intermediates, and mating to test conjugal function of plasmid.
The complete nucleotide sequence of pDYM4. 3k consisted of 4346 bp, encoding 3 genes of which one resembled Streptomyces major conjugative gene tra and other two were with unknown functions. Experiments demonstrated that a new gene of pDYM4. 3k and its upstream c. 300 bp were required for plasmid replication. Single-stranded pDYM4. 3k as a replication intermediate was detected, indicating it rolling-cirle replication mode. pDYM4.3k could conjugal transfer among Streptomyces lividans.
Streptomyces small plasmid pDYM4. 3k was able to autonomous replication and conjugal transferring.
[Show abstract][Hide abstract] ABSTRACT: Streptomyces coelicolor, with its 8 667 507-bp linear chromosome, is the genetically most studied Streptomyces species and is an excellent model for studying antibiotic production and cell differentiation. Here, we report construction of S. coelicolor derivatives containing sequential deletions of all the 10 polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters and a 900-kb subtelomeric sequence (total c. 1.22 Mb, 14% of the genome). No obvious differences in growth rates and sporulation of the strains were found. An artificially circularized S. coelicolor genome with deletions of total c. 1.6 Mb segments (840-kb for the left and 761-kb for the right arm of the linear chromosome) was obtained. The actinorhodin biosynthetic gene cluster could be overexpressed in some of the constructed strains.
Full-text · Article · Jun 2012 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.
No preview · Article · Jun 2012 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Autonomous-replicating plasmid pFP4 of Streptomyces sp. FR1 isolated from a heavy metal-contaminated land was cloned and sequenced. Surprisingly, the 40,949-bp pFP4 contains a cluster of 20 genes, resembling these chromosome-integrated prophages of Streptomyces sp. SPB78 and Streptomyces scabiei 87.22. Plasmid pFP4 could transfer by conjugation and a replication locus, iteron/repA/repB, was identified. The filtered FR1 culture could infect both FR1 and FR1 cured of pFP4 to form plaques, and also six out of 13 strains from the same land, but failed to form plaques on other seven strains from same source and all ten Streptomyces species from different sources. pFP4 phage particles were observed by transmission electron microscopy. Major structural proteins (capsid, portal and tail, etc.) of pFP4 virions were encoded by twelve pFP4 genes. pFP4 phage DNA contained 3' protruding cohesive ends of 9-nt. Streptomyces pFP4 represents a novel plasmid-phage.
[Show abstract][Hide abstract] ABSTRACT: Actinomycete species from many genera often harbor linear plasmids and some contain linear chromosomes. A self-ligation and PCR-sequencing method was developed for identifying three novel telomere sequences of linear plasmids of Streptomyces and Mycobacterium. This and four previously described methods for actinomycetes telomere cloning and sequencing are discussed.
[Show abstract][Hide abstract] ABSTRACT: Amycolatopsis mediterranei produces an important antibiotic rifamycin, the biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B. In this study, we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was able to convert rifamycin SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, but not in Streptomyce coelicolor or Mycobacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded by the rif cluster should be involved in this process.
No preview · Article · Dec 2011 · Acta Biochimica et Biophysica Sinica
[Show abstract][Hide abstract] ABSTRACT: Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains.
We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts.
We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.
[Show abstract][Hide abstract] ABSTRACT: The genus of Nocardiopsis is a new source of antibiotics, chemicals, and enzymes. Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation. By screening about 80 Nocardiopsis strains, 6 of them harbored 8 plasmids (18-80 kb). The complete nucleotide sequence of pSQ10 consisted of 18,219 bp, with 71.9% G + C content, encoding 17 open reading frames, 5 of them resembled those of Streptomyces plasmids. A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp. YIM 90083, and rep protein bound to its iteron sequence. This system may be useful for gene cloning and expression in Nocardiopsis.
[Show abstract][Hide abstract] ABSTRACT: The complete nucleotide sequence including the novel telomere sequence of Streptomyces linear plasmid pSHK1 consists of 187,263-bp, 158 genes, in which 51 genes resemble those of the linear plasmid SCP1 of Streptomyces coelicolor A3(2), and 20 genes encode transposases. Strikingly, the repetitive CRISPRs (clustered regularly interspaced short palindromic repeats) and cas (CRISPR-associated) genes were found, including a cluster of eight cas genes, in the order cas2B-cas1B-cas3B-cas5-cas4-cas2A-cas1A-cas3A, bracketed by a pair of divergent CRISPRs, and five other dispersed CRISPRs. The cas2B-cas1B-cas3B-cas5 or cas4-cas2A-cas1A genes were co-transcribed. Protein-protein interactions between Cas5 and Cas1A, 2A, 2B, 3B were detected by yeast two-hybrids, indicating a critical role of Cas5 for the formation of protein complexes. By polymerase chain reaction and Southern hybridization, 12 cas4 genes including three on linear plasmids were found among 75 newly isolated Streptomyces strains. The paired-CRISPRs and bracketed cas were also conserved in several other Streptomyces or actinomycete species. However, unlike other bacteria, the CRISPRs-cas in pSHK1 could not provide immunity against introduction of phage ΦC31 and plasmid containing the particular spacers in Streptomyces.
Preview · Article · Jun 2011 · Acta Biochimica et Biophysica Sinica