Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

Kim, K.S.; Ryu, Dewey D. Y.; Lee, S.Y.

doi:10.1016/0141-0229(83)90077-7

Cited by 14 publications

(3 citation statements)

References 19 publications

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“…Overnight cultures of S. lividans were prepared by growing each strain tested in GER medium (18) containing 50 Rg of thiostrepton per ml. A 10-,ul portion of each culture was spotted on plates containing minimal medium (13) supplemented with 2% glucose, 50 ,ug of histidine per ml, 37 ,ug of leucine per ml, 50 pLg of thiostrepton per ml, and the indicated concentrations of kanamycin (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences

Baum¹,

Buttner²,

Lin³

et al. 1989

J Bacteriol

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We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, Pla, Plb, Plc, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters Pla, Plb, and Plc, which are coordinately induced during stationary phase in M. echinospora, were examined by deletional analysis in S. lividans. The Pla and Plb promoters were functional despite deletion of native sequences 5 and 17 base pairs upstream of each initiation site, respectively. Thus, Pla and Plb had greatly reduced upstream DNA sequence requirements compared with typical procaryotic promoters. In contrast, transcription from promoter Plc was significantly decreased when native sequences 34 base pairs upstream were replaced.We have been studying promoter structure in the actinomycete Micromonospora echinospora (NRRL 15839), which grows as multicellular mycelia and has the capacity to form spores after the growing phase. During the stationary phase, M. echinospora produces the calicheamicins (19), a novel family of antitumor antibiotics that causes site-specific double-stranded cleavage of DNA (29).Previously, we defined a set of multiple tandem promoters within a 0.4-kilobase (kb) DNA fragment that has several novel features (1). The P1 promoter region consists of three transcriptional start sites separated by 12 and 17 nucleotides (nt), which are maximally active during stationary phase, concurrent with calicheamicin production. Promoter P2 is located 80 base pairs (bp) downstream from the P1 region and is maximally active during the exponential phase of the life cycle. The P1 and P2 promoters are potentially useful for the investigation of gene activation in actinomycetes during the transition from exponential to stationary phase, when many secondary metabolites are produced (22).The P1 and P2 promoters, and an additional site designated Ptk, are recognized by Streptomyces lividans transformants harboring the 0.4-kb fragment on a plasmid (1). We have therefore utilized the well-characterized genetic system of Streptomyces for more detailed transcriptional analysis. The P1 and P2 promoters belong to the major class of Streptomyces promoters that do not function in Escherichia coli (12,15,16). A consensus sequence has not been identified for this class of promoters. The occurrence of multiple forms of RNA polymerase holoenzyme in Streptomyces spp. which direct transcription from different promoters (6, 28) is consistent with the diversity of promoter sequences found in this organism. Recently, four different genes apparently encoding sigma factors were identified in S. coelicolor by homology to sequences derived from E. coli and Bacillus subtilis (26). In addition, the whiG locus also encodes a putative sigma facto...

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Section: Methodsmentioning

confidence: 99%

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences

Baum¹,

Buttner²,

Lin³

et al. 1989

J Bacteriol

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“…The protoplast suspension was centrifuged, diluted, and then stained with crystal violet (Savitha et al, 2010). The numbers of protoplasts were determined using a current formula (cells/ml) = the average count per square × the dilution factor × 10 4 (Kim, Ryu, & Lee, 1983).…”

Section: Examination Of Protoplastmentioning

confidence: 99%

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

Hirpara

Gajera

Patel

et al. 2018

Journal Cellular Physiology

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The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management. K E Y W O R D S inhibition coefficient, molecular heterozygosity, protoplast fusion, Sclerotium rolfsii, Trichoderma

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“…Transformation of Streptomyces protoplasts with plasmid DNA has been well documented (4). Procedures have been described for protoplasting and regenerating Micromonospora species (7,8,12,13,(15)(16)(17). Successful transformation of Micromonospora sp., however, is rare.…”

mentioning

confidence: 99%

Conditions for protoplasting, regenerating, and transforming the calicheamicin producer, Micromonospora echinospora

Love

Maiese

Rothstein

1992

Appl Environ Microbiol

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Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

Cited by 14 publications

References 19 publications

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

Conditions for protoplasting, regenerating, and transforming the calicheamicin producer, Micromonospora echinospora

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