New derivatives of the Streptomyces temperate phage φC31 useful for the cloning and functional analysis of Streptomyces DNA

Rodicio, M. Rosario; Bruton, Celia J.; Chater, Keith F.

doi:10.1016/0378-1119(85)90137-4

Cited by 73 publications

(47 citation statements)

References 13 publications

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“…In most cases, however, the cloned tyr genes have somewhat complex regulation of their own that complicates their use as reporter genes. Drug resistance genes have been used successfully in promoter-probe vectors and have allowed the identification of many Streptomyces promoters (4,30,38); however, their use requires selectable levels of expression during vegetative growth, and this limits their use for the study of developmentally regulated promoters. Horinouchi and Beppu (20) constructed a vector based on a gene from S. coelicolor that encodes a brown compound, but its use requires special hosts or growth conditions in which little or no interfering pigment is produced.…”

Section: Resultsmentioning

confidence: 99%

“…Several other reporter gene systems have been investigated for use in Streptomyces spp. These include systems based on the E. coli ampC gene (a ,-lactamase) (15); the Streptomyces glaucescens tyr gene (a tyrosinase) (18,20); the E. coli galK (galactokinase) (8,31), neo (an aminoglycoside phosphotransferase from Tn5) (4,38), and cat (a chloramphenicol acetyltransferase) (4) genes; and the Streptomyces vinaceus vph gene (a phosphotransferase) (30). Although each of these has proved useful in some applications, all have disadvantages; most lack the sensitivity of lacZ (in E. coli) for detection of gene activity in colonies on plates, and there is no evidence that they accurately reflect the activity of promoters driving their expression.…”

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confidence: 99%

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xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter

et al. 1989

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We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp. based on the use of a promoterless copy of the xylE gene of Pseudomonas putida. The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde. A promoterless copy of xyIE was placed under the transcriptional control of galPI, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source. On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation. Activity of gaUlPI-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots. Moreover, differences in expression levels of xylE fusions driven by mutant galPI promoters were readily apparent in color reactions on plates. The properties ofxylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp. but also for recovering mutations that alter the expression levels of promoters of interest.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter

et al. 1989

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“…Candida utilis (synonym, Pichia jadinii) CECT 1061 was used for bioassay experiments. Phage KC515 (c ϩ attP::tsr::vph), a C31-derived phage (38), was used for gene disruption experiments. Streptomyces lividans JII 1326 (17) served as a host for phage propagation and transfection.…”

Section: Methodsmentioning

confidence: 99%

Identification of PimR as a Positive Regulator of Pimaricin Biosynthesis in Streptomyces natalensis

et al. 2004

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“…The Streptomyces plasmid vector was the high-copy-number pIJ702 (21). The Streptomyces phage vector was the C31-derivative KC515 (43).…”

Section: Methodsmentioning

confidence: 99%

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation

1997

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In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gramnegative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein.Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.During a screening process, a streptomycete producer of broad-range antibiotic activity was isolated from soil samples; this strain has been taxonomically characterized as Streptomyces rochei F20, and its active compound has been described as a mixture of streptothricins (mostly F and traces of D and E) (41a). The present paper describes the isolation, DNA sequence, and partial characterization of the biosynthetic pathway for streptothricin produced by this newly isolated strain. Streptothricin was one of the first actinomycete antibiotics to be described (56). It has a broad spectrum, with antibacterial as well as antifungal activity. Its chemical elucidation was described by Kusumoto and coworkers (30) (Fig. 1). It contains a heterocyclic ␤-amino acid (streptolidine), an amino sugar (4-carbamido-D-gulosamine), and, by amide linkage at C-2, a ␤-lysine chain which varies from one to six units in streptothricins F to A, respectively, and which includes seven units for streptothricin X (22, 31). More recently, other members of the family, which are chemically closely related to streptothricin, have been described (1,23,25).Members of the streptothricin family have, in addition to a potent inhibitory activity for prokaryotic protein synthesis (11), cytotoxicity which prevents their clinical or veterinary use. However, this may well be useful in situations in wh...

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New derivatives of the Streptomyces temperate phage φC31 useful for the cloning and functional analysis of Streptomyces DNA

Cited by 73 publications

References 13 publications

xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter

xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter

Identification of PimR as a Positive Regulator of Pimaricin Biosynthesis in Streptomyces natalensis

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation

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