Nejvíce citovaný článek - PubMed ID 3002927
Transformation of Streptomyces granaticolor with natural and recombinant plasmid vectors
A time-correlated expression of eukaryotic-like protein Ser/Thr kinase Pkg2 of Streptomyces granaticolor was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by transcriptional fusion experiments. In a complex medium the activity of pkg2 promoter was constant during the life cycle. Direct RNA analysis proved the presence of corresponding pkg2 transcript. S1 nuclease protection analysis of the transcription initiation site showed that pkg2 gene is expressed as a leaderless mRNA. Under phosphate starvation the promoter activity was detectable merely in the early exponential phase. Under these conditions turning off of pkg2 promoter and cessation of pkg2 transcript level coincided with the start of granaticin production.
- MeSH
- fosfáty metabolismus MeSH
- genetická transkripce MeSH
- katechol-2,3-dioxygenasa metabolismus MeSH
- kultivační média MeSH
- promotorové oblasti (genetika) MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- Streptomyces enzymologie genetika růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfáty MeSH
- katechol-2,3-dioxygenasa MeSH
- kultivační média MeSH
- protein-serin-threoninkinasy MeSH
A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
- MeSH
- bakteriální geny MeSH
- DNA bakterií genetika MeSH
- DNA primery genetika MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fenotyp MeSH
- fosforylace MeSH
- klonování DNA MeSH
- mikroskopie elektronová rastrovací MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- protein-serin-threoninkinasy chemie genetika metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- restrikční mapování MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- Streptomyces enzymologie genetika ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- DNA primery MeSH
- protein-serin-threoninkinasy MeSH
- rekombinantní fúzní proteiny MeSH
A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.
