Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer

Qin, Zhongjun; Peng, Kaiman; Zhou, Xing; Liang, Rongfang; Zhou, Qi; Chen, Huakui; Hopwood, David A.; Kieser, Tobias; Deng, Zixin

doi:10.1128/jb.176.7.2090-2095.1994

Cited by 19 publications

(29 citation statements)

References 20 publications

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“…Interestingly, three putative regulatory genes were identified, with two at the end of, and one flanking, the cluster. Since S. hygroscopicus 10-22 is much more difficult to manipulate genetically than S. hygroscopicus 5008 (Pang et al, 2002;Qin et al, 1994), here we used strain 5008 as a model to investigate the roles of these three regulatory genes in the biosynthesis of cyclothiazomycin. We proved that the cluster is functional in S. hygroscopicus 5008 and we showed that one of the regulatory genes, SHJG8833, is essential for cyclothiazomycin biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

Zhang

Chen

et al. 2014

Microbiology

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Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the clt cluster encoding cyclothiazomycin from Streptomyces hygroscopicus 10-22 was revealed by genome sequencing in S. hygroscopicus 5008. Genes SHJG8833 and SHJG8837 of the cluster and flanking gene SHJG8838 were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that SHJG8833, but not SHJG8837 or SHJG8838, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of SHJG8833 was located to a thymidine 54 nt upstream of the start codon. Inactivation of SHJG8833 abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing SHJG8833 into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from SHJG8826 to SHJG8832, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons, SHJG8826-27 and SHJG8828-32. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of SHJG8826 and SHJG8828 and sequences internal to SHJG8826 and SHJG8829, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the clt cluster.

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Section: Introductionmentioning

confidence: 99%

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

Zhang

Chen

et al. 2014

Microbiology

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“…A 1-kb supercoiled DNA ladder was used as a size marker. a pSLA2 circular plasmids were introduced into ZX7, and thiostrepton-resistant colonies were inoculated into CM medium containing thiostrepton (23). After 7 days at 30°C, spores were streaked on CM plates without selection for 7 days at the same temperature.…”

Section: Discussionmentioning

confidence: 99%

“…Culture and transformation of Streptomyces followed the methods of Kieser et al (16). To obtain good sporulation, strain ZX7 grew on complete medium (23). Plasmid pLUS450, kindly provided by Carton Chen, contained 2.6-kb chromosomal telomere sequences.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a pSLA2 Plasmid Locus Required for Linear DNA Replication and Circular Plasmid Stable Inheritance in Streptomyces lividans

2003

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Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.Streptomyces species are among the few eubacteria known to include both linear chromosomes and linear plasmids (10,17,20). The telomeres of Streptomyces linear replicons contain a series of short inverted repeat DNA sequences (7,9,12,18,22) and are capped by terminal proteins linked covalently to 5Ј ends of double-stranded DNA (1,9,20,33). Unlike adenovirus and bacteriophage 29, which also have terminal protein linked covalently to 5Ј DNA ends but which replicate by a strand displacement mechanism (25), Streptomyces linear plasmids contain a centrally located origin and replicate bidirectionally (4). This process has been shown to leave 280 nucleotides of single-stranded DNA at the 3Ј ends of pSLA2 replication intermediates; these are then filled in (4, 5), possibly by a fold-back mechanism involving the inverted repeats of telomeres (22). As Streptomyces chromosomes also appear to duplicate their genes bidirectionally (21) and Streptomyces coelicolor and Streptomyces lividans chromosomal telomeres are highly similar to those of pSLA2 (12,22), the filling in of recessed 5Ј ends of linear chromosomes is presumed to occur by a similar mechanism.Streptomyces linear replicons can replicate in both linear and circular form when their telomeres are deleted (4,19,20,27,30). The site of initiation of DNA replication for Streptomyces linear plasmid pSLA2 has been identified experimentally (4) and found to include a region containing short direct repeats (iterons) as well as a DNA helicas...

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“…Microbiological and genetic manipulations in Escherichia coli and Streptomyces were according to Kieser et al (2000). Five solid media were used for culturing Streptomyces strains: NE (per litre: 10 g glucose, 2 g yeast extract, 2 g Casamino acids, 1 g beef extract, 15 g agar, pH 7.0) (Thiara & Cundliffe, 1995), PYM (per litre: 5 g peptone, 3 g yeast extract, 3 g malt extract, 10 g glucose, 20 g agar), LB, R5 (Kieser et al, 2000) and HAUCM (Qin et al, 1994). Dot-blot hybridization.…”

Section: Methodsmentioning

confidence: 99%

A multidrug efflux system is involved in colony growth in Streptomyces lividans

et al. 2007

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Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer

Cited by 19 publications

References 20 publications

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

Identification and Characterization of a pSLA2 Plasmid Locus Required for Linear DNA Replication and Circular Plasmid Stable Inheritance in Streptomyces lividans

A multidrug efflux system is involved in colony growth in Streptomyces lividans

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