The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S. aureofaciens CCM 3239 strain to overcome this strict regulation. A regulatory region including aur1R, aur1P, aur1O and the target biosynthetic aur1Ap promoter were replaced by the strong constitutive ermEp* promoter. However, auricin production was decreased in such a genetically manipulated strain. In the second strategy we placed the aur1P gene for auricin pathway-specific activator under the control of the ermEp* promoter. The resulting strain has been shown to produce 2.8-fold higher amount of auricin compared with the WT strain.
- MeSH
- antibakteriální látky biosyntéza MeSH
- DNA bakterií genetika metabolismus MeSH
- makrolidy metabolismus MeSH
- multigenová rodina MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- restrikční mapování MeSH
- Streptomyces aureofaciens genetika metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.
Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.
- MeSH
- bakteriální proteiny MeSH
- deoxyribonukleasy MeSH
- exonukleasy MeSH
- financování organizované MeSH
- fosfatasy MeSH
- klonování DNA MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- Streptomyces aureofaciens enzymologie genetika chemie MeSH
- Streptomyces coelicolor enzymologie genetika chemie MeSH
- substrátová specifita MeSH
- umlčování genů MeSH
tmRNA and protein SmpB are the main components required for rescue of stalled ribosomes incapable of properly elongating or terminating the polypeptide chain. We examined the tmRNA level and protein synthesis in Streptomyces aureofaciens, S. griseus and S. collinus synthesizing tetracycline, streptomycin and kirromycin, respectively, during various stress conditions. Downshift in temperature caused a decrease in protein synthesis but the level of tmRNA increased. Shift up in temperature induced decay of tmRNA in all strains and in S. collinus led to stimulation and in S. aureofaciens and S. griseus to inhibition of protein synthesis. At high NaCl concentrations protein synthesis was inhibited and tmRNA decayed. Shift in pH from 7.0 to 5.0 had no pronounced effect on the tmRNA level while upshift to pH 9.0 in S. collinus and S. aureofaciens caused inhibition of protein synthesis and decay of tmRNA in S. collinus. In contrast, protein synthesis and tmRNA level increased in S. griseus at the alkaline pH. Our data show that tmRNA abundance is important for survival of streptomycetes under certain unfavorable conditions.
- MeSH
- bakteriální RNA analýza genetika MeSH
- elektroforéza v polyakrylamidovém gelu metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- hybridizace genetická genetika MeSH
- northern blotting metody využití MeSH
- proteosyntéza genetika MeSH
- reakce na tepelný šok genetika MeSH
- Streptomyces aureofaciens genetika metabolismus MeSH
- Streptomyces griseus genetika metabolismus MeSH
In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- fosforylace MeSH
- proteosyntéza genetika MeSH
- spektroskopie infračervená s Fourierovou transformací metody využití MeSH
- Streptomyces aureofaciens genetika MeSH
- Streptomyces coelicolor genetika MeSH
- western blotting metody využití MeSH
The entire double-stranded DNA genome of the Streptomyces aureofaciens phage mu1/6 was sequenced and analyzed. Its size is 38.194 kbp with an overall molar G+C content of 71.2 %. Fifty-two potential open reading frames (orfs) were identified, divided into two oppositely transcribed regions. In the left arm of the mu1/6 genome, an identified putative integrase and possible regulation proteins were identified. The rightwards transcribed region contains genes organized into apparently four functional units responsible for: (i) replication, (ii) DNA packaging and head assembly, (iii) tail morphogenesis, and (iv) lysis. Putative functions were assigned to twelve orfs based on bioinformatic analysis or experimental substantiation. Comparative analysis with three complete genomes of streptomycete phages revealed resemblance with respect to the organization of their genes into functional modules. Closer relationship was observed only between mu1/6 and S. venezuelae phage VWB.
- MeSH
- bakteriofágy genetika imunologie MeSH
- finanční podpora výzkumu jako téma MeSH
- genomová knihovna MeSH
- komponenty genomu genetika MeSH
- lyzogenie genetika MeSH
- otevřené čtecí rámce genetika MeSH
- Siphoviridae genetika izolace a purifikace MeSH
- Streptomyces aureofaciens genetika izolace a purifikace MeSH
- tetracyklin farmakologie izolace a purifikace MeSH
- MeSH
- chloramfenikol biosyntéza MeSH
- financování vládou MeSH
- messenger RNA biosyntéza genetika MeSH
- polymerázová řetězová reakce metody využití MeSH
- proteosyntéza genetika MeSH
- RNA transferová biosyntéza genetika MeSH
- Streptomyces aureofaciens genetika izolace a purifikace růst a vývoj MeSH
- tetracyklin MeSH
- Publikační typ
- techniky in vitro MeSH
