The glass beads cultivation system developed in our laboratory for physiological studies of filamentous microorganisms supports differentiation and allows complete recovery of bacterial colonies and their natural products from cultivation plates. Here, we used this system to study the global effect of ppk gene disruption in Streptomyces lividans. The ppk encoding the enzyme polyphosphate kinase (P) catalyses the reversible polymerisation of gamma phosphate of ATP to polyphosphates. The resulting are phosphate and energy stock polymers. Because P activity impacts the overall energetic state of the cell, it is also connected to secondary metabolite (e.g. antibiotic) biosynthesis. We analysed the global effects of the disruption of this gene including its influence on the production of pigmented antibiotics, on morphological differentiation, on the levels of ATP and on the whole cytoplasmic protein expression pattern of S. lividans. We observed that the S. lividans ppk mutant produced antibiotics earlier and in greater amount than the wild-type (wt) strain. On the other hand, we did not observe any obvious effect on colony morphological development. In agreement with the function of Ppk, we detected much lower levels of ATP in ppk- mutant than in the wt strain. Proteomic analysis revealed that the genes that were influenced by ppk inactivation included enzymes involved in carbon or nitrogen metabolism, phosphate transport and components of the cell translational machinery. We showed that the synthesis of translation elongation factor Tu is during sporulation much higher in ppk- mutant than in wild-type strain.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• antibakteriální látky biosyntéza   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fosfotransferasy s fosfátovou skupinou jako akceptorem genetika   metabolismus   MeSH
• kultivační techniky přístrojové vybavení   metody   MeSH
• molekulární sekvence - údaje MeSH
• regulace genové exprese u bakterií MeSH
• Streptomyces lividans enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• umlčování genů * MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• fosfotransferasy s fosfátovou skupinou jako akceptorem MeSH
• polyphosphate kinase MeSH Prohlížeč

We present the results of analysis of membrane phosphoproteomes from individual morphological stages of Streptomyces coelicolor that reflect developmentally dependent heterogeneity and phosphorylation of intrinsic and externally added purified Strepomyces aureofaciens EF-Tu. Fast growing nonpathogenic Mycobacterium smegmatis was used as a non-differentiating actinomycetes comparative model. Streptomycetes membrane fraction was found to contain protein kinase(s) catalyzing phosphorylation of both its own and an externally added EF-Tu, whereas Mycobacterium membrane fraction contains protein kinase phosphorylating only its own EF-Tu.

• MeSH
• buněčná membrána chemie   enzymologie   metabolismus   MeSH
• elongační faktor Tu izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• Mycobacterium smegmatis chemie   enzymologie   metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• proteinkinasy izolace a purifikace   metabolismus   MeSH
• Streptomyces chemie   enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• elongační faktor Tu MeSH
• proteinkinasy MeSH

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny analýza   genetika   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• luciferasy bakteriální analýza   genetika   MeSH
• methyltransferasy genetika   MeSH
• mutageneze cílená MeSH
• nesmyslný kodon MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• proteosyntéza * MeSH
• reportérové geny * MeSH
• Streptomyces lividans účinky léků   genetika   fyziologie   MeSH
• streptomycin farmakologie   MeSH
• supresorové geny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• inhibitory syntézy proteinů MeSH
• luciferasy bakteriální MeSH
• methyltransferasy MeSH
• nesmyslný kodon MeSH
• rRNA (adenosine-O-2'-)methyltransferase MeSH Prohlížeč
• streptomycin MeSH

A two-phase cultivation system was developed which will enable studies of streptomycete differentiation by molecular biological and global techniques such as transcriptomics and proteomics. The system is based on a solid phase formed by glass beads corresponding to particles in soil, clay, or sand natural habitats of streptomycetes. The beads are immersed in a liquid medium that allows easy modification or replacement of nutrients and growth factors as well as radioactive labeling of proteins. Scanning electron microscopy was used to analyze morphological differentiation of streptomycetes on glass beads and two-dimensional protein electrophoresis to demonstrate the potential of the system for analyses of protein synthesis profiles during the developmental program. This system facilitates studies of differentiation including expression and post-translation modifications of streptomycetes proteins, secondary metabolite biosynthesis, and morphological development.

• MeSH
• 2D gelová elektroforéza MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriologické techniky MeSH
• kultivační média MeSH
• mikroskopie elektronová rastrovací MeSH
• proteomika MeSH
• regulace genové exprese u bakterií MeSH
• sklo * MeSH
• Streptomyces růst a vývoj   metabolismus   ultrastruktura   MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• kultivační média MeSH