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
• Adenosine Triphosphate biosynthesis   MeSH
• Anti-Bacterial Agents biosynthesis   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Phosphotransferases (Phosphate Group Acceptor) genetics   metabolism   MeSH
• Culture Techniques instrumentation   methods   MeSH
• Molecular Sequence Data MeSH
• Gene Expression Regulation, Bacterial MeSH
• Streptomyces lividans enzymology   genetics   growth & development   metabolism   MeSH
• Gene Silencing * MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Phosphotransferases (Phosphate Group Acceptor) MeSH
• polyphosphate kinase MeSH Browser

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
• Cell Membrane chemistry   enzymology   metabolism   MeSH
• Peptide Elongation Factor Tu isolation & purification   metabolism   MeSH
• Phosphorylation MeSH
• Mycobacterium smegmatis chemistry   enzymology   metabolism   MeSH
• Protein Processing, Post-Translational * MeSH
• Protein Kinases isolation & purification   metabolism   MeSH
• Streptomyces chemistry   enzymology   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Peptide Elongation Factor Tu MeSH
• Protein Kinases 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
• Electrophoresis, Gel, Two-Dimensional MeSH
• Peptide Elongation Factor Tu chemistry   isolation & purification   metabolism   MeSH
• Phosphorylation MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Protein Kinases metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Spores, Bacterial enzymology   metabolism   MeSH
• Streptomyces aureofaciens metabolism   MeSH
• Streptomyces coelicolor metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Imaging, Three-Dimensional MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptide Elongation Factor Tu MeSH
• Protein Kinases MeSH

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Bacterial Proteins genetics   immunology   isolation & purification   metabolism   MeSH
• Inclusion Bodies ultrastructure   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Peptide Elongation Factor Tu genetics   immunology   isolation & purification   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression MeSH
• Genetic Vectors MeSH
• Guanosine Diphosphate metabolism   MeSH
• Cloning, Molecular MeSH
• Microscopy, Electron, Scanning MeSH
• Antibodies, Monoclonal immunology   MeSH
• Plasmids MeSH
• Protein Processing, Post-Translational * MeSH
• Protein Isoforms immunology   MeSH
• Recombinant Proteins immunology   isolation & purification   metabolism   MeSH
• Streptomyces aureofaciens genetics   MeSH
• Microscopy, Electron, Transmission MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Peptide Elongation Factor Tu MeSH
• Guanosine Diphosphate MeSH
• Antibodies, Monoclonal MeSH
• Protein Isoforms MeSH
• Recombinant Proteins MeSH

An asporogenous spontaneous mutant of Streptomyces aureofaciens named ASR1 was selected on streptomycin gradient plates. The mutant is very stable and differs in ultrastructure and morphology, it is prototrophic but it lost the ability to grow well on soybean extract medium and produces one-tenth tetracyclines of the parent. The ASR1 mutant has a 3-4-fold increased resistance to streptomycin and is cross-resistant to other aminoglycosides. Comparison of the protein profiles from both strains on SDS gels revealed a very low expression of a 29.5 kDa protein in the ASR1 mutant which is overexpressed in both vegetative cells and spores of the parental strain.

• MeSH
• Aminoglycosides MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Microbial genetics   MeSH
• Bacterial Proteins genetics   isolation & purification   MeSH
• Mutation MeSH
• Spores, Bacterial genetics   MeSH
• Streptomyces aureofaciens drug effects   genetics   ultrastructure   MeSH
• Tetracycline biosynthesis   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aminoglycosides MeSH
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Tetracycline MeSH