BACKGROUND: The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. RESULTS: Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. CONCLUSIONS: Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.

• MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální RNA genetika   MeSH
• buněčné linie MeSH
• delece genu * MeSH
• fenotyp MeSH
• makrofágy mikrobiologie   MeSH
• mikrobiální viabilita MeSH
• myši MeSH
• oligopeptidy biosyntéza   MeSH
• oxidační stres * MeSH
• protein-serin-threoninkinasy genetika   MeSH
• Pseudomonas aeruginosa genetika   růst a vývoj   patogenita   MeSH
• regulace genové exprese u bakterií MeSH
• salát (hlávkový) mikrobiologie   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• testy genetické komplementace MeSH
• transkriptom MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• oligopeptidy MeSH
• ppkA protein, Pseudomonas aeruginosa MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• pyoverdin MeSH Prohlížeč

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.

• MeSH
• akademie a ústavy dějiny   MeSH
• dějiny 20. století MeSH
• mikrobiální genetika dějiny   MeSH
• molekulární biologie dějiny   MeSH
• Check Tag
• dějiny 20. století MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Česká republika MeSH

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

In vitro phosphorylation reaction using extracts prepared from cells in the exponential phase of growth and aerial spores of Streptomyces coelicolor displayed the presence of multiply phosphorylated proteins. Effect of protein kinase inhibitors (PKIs) (geldanamycin, wortmannin, apigenin, genistein, roscovitine, methyl 2,5-dihydroxycinnamate, rapamycin, staurosporine) was determined on protein phosphorylation and on germination of spores. The in vitro experiments showed differences in phosphoprotein pattern due to the presence of PKIs. Cultivation of aerial spores with PKIs led to a significant delay in germ tube emergence and filament formation. However, none of the tested PKIs completely blocked the germination process. These results indicate that protein kinases of spores form complex networks sharing common modulating site that plays an important role in proper timing of early developmental events.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• fosforylace účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• proteinkinasy účinky léků   metabolismus   MeSH
• spory bakteriální účinky léků   metabolismus   MeSH
• Streptomyces coelicolor účinky léků   enzymologie   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• inhibitory proteinkinas MeSH
• proteinkinasy MeSH

Signal transduction pathways in both prokaryotes and eukaryotes utilize protein phosphorylation as a key regulatory mechanism. Recent studies have proven that eukaryotic-type serine/threonine protein kinases (Hank's type) are widespread in many bacteria, although little is known regarding the cellular processes they control. In this study, we have attempted to establish the role of a single eukaryotic-type protein kinase, StkP of Streptococcus pneumoniae, in bacterial survival. Our results indicate that the expression of StkP is important for the resistance of S. pneumoniae to various stress conditions. To investigate the impact of StkP on this phenotype, we compared the whole-genome expression profiles of the wild-type and DeltastkP mutant strains by microarray technology. This analysis revealed that StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response. Despite the reduced transformability of the stkP mutant, we found that the competence regulon was derepressed in the stkP mutant under conditions that normally repress natural competence development. Furthermore, the competence regulon was expressed independently of exogenous competence-stimulating peptide. In summary, our studies show that a eukaryotic-type serine/threonine protein kinase functions as a global regulator of gene expression in S. pneumoniae.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• delece genu MeSH
• eukaryotické buňky enzymologie   MeSH
• fenotyp MeSH
• koncentrace vodíkových iontů MeSH
• mikrobiální viabilita účinky léků   genetika   MeSH
• mutace MeSH
• osmotický tlak MeSH
• oxidační stres MeSH
• peroxid vodíku farmakologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• Streptococcus pneumoniae enzymologie   genetika   růst a vývoj   MeSH
• testy genetické komplementace MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• peroxid vodíku MeSH
• protein-serin-threoninkinasy MeSH

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

The existence of phosphoprotein phosphatase (PPP) in aerial mycelium of S. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfoserin metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• proteinfosfatasy analýza   antagonisté a inhibitory   metabolismus   MeSH
• Streptomyces enzymologie   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoserin MeSH
• fosfothreonin MeSH
• inhibitory enzymů MeSH
• proteinfosfatasy MeSH