In this paper, correlation analysis of protein and mRNA levels in the soil dwelling bacteria Streptomyces coelicolor (S. coelicolor M145) is presented during development of the population as it grew in liquid medium using three biological and two technical replicates, measured during exponential growth, and its entry into the stationary phase. The proteome synthesis time series are compared with the gene expression time series measured previously under identical experimental conditions. Results reveal that about one third of protein/mRNA synthesis profiles are well correlated while another third are correlated negatively. Functional analysis of the highly correlated groups is presented. Based on numerical simulation, the negative correlation between protein and mRNA is shown to be caused by the difference between the rate of translation and protein degradation.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- proteom analýza metabolismus MeSH
- půda chemie MeSH
- regulace genové exprese u bakterií MeSH
- stanovení celkové genové exprese MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- transkriptom * MeSH
- vývojová regulace genové exprese * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.
- MeSH
- anthrachinony metabolismus MeSH
- antibakteriální látky metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- kultivační média metabolismus MeSH
- sekundární metabolismus MeSH
- sírany metabolismus MeSH
- Streptomyces coelicolor enzymologie genetika růst a vývoj metabolismus MeSH
- thiosulfátsulfurtransferasa genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCE: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- delece genu MeSH
- molekulární sekvence - údaje MeSH
- posttranslační úpravy proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Bacterial spore germination is a developmental process during which all required metabolic pathways are restored to transfer cells from their dormant state into vegetative growth. Streptomyces are soil dwelling filamentous bacteria with complex life cycle, studied mostly for they ability to synthesize secondary metabolites including antibiotics. RESULTS: Here, we present a systematic approach that analyzes gene expression data obtained from 13 time points taken over 5.5 h of Streptomyces germination. Genes whose expression was significantly enhanced/diminished during the time-course were identified, and classified to metabolic and regulatory pathways. The classification into metabolic pathways revealed timing of the activation of specific pathways during the course of germination. The analysis also identified remarkable changes in the expression of specific sigma factors over the course of germination. Based on our knowledge of the targets of these factors, we speculate on their possible roles during germination. Among the factors whose expression was enhanced during the initial part of germination, SigE is though to manage cell wall reconstruction, SigR controls protein re-aggregation, and others (SigH, SigB, SigI, SigJ) control osmotic and oxidative stress responses. CONCLUSIONS: From the results, we conclude that most of the metabolic pathway mRNAs required for the initial phases of germination were synthesized during the sporulation process and stably conserved in the spore. After rehydration in growth medium, the stored mRNAs are being degraded and resynthesized during first hour. From the analysis of sigma factors we conclude that conditions favoring germination evoke stress-like cell responses.
The aim of this study was to contribute to clarifying the role of 6S RNA in the development and control of antibiotic biosynthesis in Streptomyces coelicolor. Due to the low energetic cost of gene silencing via 6S RNA, it is an easy and rapid means of down-regulating the expression of specific genes in response to signals from changes in the environment. The expression of 6S RNA in S. coelicolor is not constitutive, and its accumulation is adapted to changes in nutritional conditions. The 6S RNA of S. coelicolor is capable of interacting with RNA polymerase β β' subunits and is a template for the transcription of short pRNAs. Deletion of the ssrS gene from S. coelicolor affects the growth rate and causes changes in the expression of several pathway-specific genes involved in actinorhodin biosynthesis. The complementation of the ΔssrS strain with ssrS gene restored the wild-type levels of growth and actinorhodin production. We conclude that 6S RNA contributes to the optimization of cellular adaptation and is an important factor involved in the regulation of growth and expression of key genes for the biosynthesis of actinorhodin.
- MeSH
- antibakteriální látky biosyntéza MeSH
- bakteriální RNA genetika metabolismus MeSH
- delece genu MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- nekódující RNA genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- testy genetické komplementace MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An example of bacterium, which undergoes a complex development, is the genus of Streptomyces whose importance lies in their wide capacity to produce secondary metabolites, including antibiotics. In this work, a proteomic approach was applied to the systems study of germination as a transition from dormancy to the metabolically active stage. The protein expression levels were examined throughout the germination time course, the kinetics of the accumulated and newly synthesized proteins were clustered, and proteins detected in each group were identified. Altogether, 104 2DE gel images at 13 time points, from dormant state until 5.5 h of growth, were analyzed. The mass spectrometry identified proteins were separated into functional groups and their potential roles during germination were further assessed. The results showed that the full competence of spores to effectively undergo active metabolism is derived from the sporulation step, which facilitates the rapid initiation of global protein expression during the first 10 min of cultivation. Within the first hour, the majority of proteins were synthesized. From this stage, the full capability of regulatory mechanisms to respond to environmental cues is presumed. The obtained results might also provide a data source for further investigations of the process of germination.
- MeSH
- 2D gelová elektroforéza MeSH
- antibakteriální látky biosyntéza MeSH
- hmotnostní spektrometrie MeSH
- proteom analýza MeSH
- proteosyntéza * MeSH
- regulace genové exprese u bakterií MeSH
- spory bakteriální * růst a vývoj metabolismus MeSH
- Streptomyces coelicolor * genetika růst a vývoj metabolismus MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The genome of Streptomyces coelicolor encodes six potential WD-40 genes. Two of them, the wdpB (SCO5953) and the wdpC (SCO4422) genes, were studied to determine their function. Deletion of the wdpB gene resulted in a considerable decrease of aerial hyphae formation, leading to a conditionally bald phenotype, and reduced undecylprodigiosin production. In addition, the aerial hyphae of the ΔwdpB mutant strain were unusually branched and showed the signs of irregular septation and precocious lysis. Disruption of wdpC resulted in the reduction of undecylprodigiosin and delayed actinorhodin production. The ΔwdpC mutant strain showed precocious lysis of hyphae and delayed sporulation without typical curling of aerial hyphae in the early sporulation stage. The whole-genome transcriptome analysis revealed that deletion of wdpB affects the expression of genes involved in aerial hyphae differentiation, sporulation and secondary metabolites production. Deletion of wdpC caused downregulation of several gene clusters encoding secondary metabolites. Both the wdp genes seem to possess transcriptional autoregulatory function. Overexpression and genetic complementation studies confirmed the observed phenotype of both mutants. The results obtained suggest that both genes studied have a pleiotropic effect on physiological and morphological differentiation.
- MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika metabolismus MeSH
- delece genu MeSH
- genetická transkripce MeSH
- multigenová rodina MeSH
- regulace genové exprese u bakterií MeSH
- repetitivní sekvence aminokyselin * MeSH
- sekundární metabolismus MeSH
- stanovení celkové genové exprese MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- transkriptom MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Silent information regulators are NAD(+)-dependent enzymes that display differential specificity toward acetylated substrates. This report provides first evidence for deacetylation activity of CobB1 in Streptomyces coelicolor. The protein is highly conserved in streptomycetes. The CobB1 protein catalytically removes the acetyl group from acetylated bovine serum albumin. In the absence of NAD+ or when NAD+ was substituted with nicotinamide, deacetylation was stopped. We isolated gene encoding AcetylCoA synthetaseA. The recombinant enzyme produces Acetyl-CoA from acetate. The highest acsA-mRNA level was detected in cells from the exponential phase of growth, and then decreased in transition and stationary phases of growth. Acetylated acsA loses the ability to transfer acetate to CoA. Deacetylation of the enzyme required CobB1, ATP-Mg2, and NAD+. Using specific antibodies against acetylated lys, CobB1, and acsA, we found relationship between level of CobB1 and acetylation of acsA, indicating that CobB1 is involved in regulating the acetylation level of acsA and consequently its activity. It was found that 1-acetyl-tetrahydroxy and 1-acetyl pentahydroxy antraquinone inhibit the deacetylation activity of CobB1.
- MeSH
- acetát-CoA-ligasa biosyntéza chemie genetika MeSH
- acetylace MeSH
- anthrachinony chemie MeSH
- bakteriální proteiny biosyntéza chemie izolace a purifikace MeSH
- genetická transkripce MeSH
- inhibitory enzymů chemie MeSH
- katalytická doména MeSH
- konzervovaná sekvence MeSH
- molekulární sekvence - údaje MeSH
- posttranslační úpravy proteinů MeSH
- regulace genové exprese u bakterií MeSH
- rekombinantní proteiny antagonisté a inhibitory biosyntéza chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- sirtuiny antagonisté a inhibitory biosyntéza chemie MeSH
- Streptomyces coelicolor enzymologie růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transfer-messenger RNA (tmRNA, 10Sa RNA, ssrA) is bacterial RNA having both tRNA and mRNA properties and playing an essential role in recycling of 70S ribosomes that are stalled on defective mRNA. The trans-translational system is thought to play a crucial role in bacterial survival under adverse conditions. Streptomycetes are Gram-positive soil bacteria exposed to various physical and chemical stresses that activate specialized responses such as synthesis of antibiotics and morphological differentiation. Comparative sequence analysis of ssrA genes of streptomycetes revealed the most significant differences in the central parts of tag-reading frames, in the stop codons and in the 15-34 nucleotide sequences following stop codons. A major challenge in understanding the interactions that control the function of tmRNA is the definition of protein interactions. Proteins from various phases of development of Streptomyces aureofaciens associated with tmRNA were analyzed. Using affinity chromatography on tmRNA-Sepharose and photo cross-linking experiments with [(32)P]labeled tmRNA seven proteins, the beta and beta'-subunits of DNA dependent RNA polymerase, polyribonucleotide nucleotidyltransferase (PNPase), ribosomal protein SS1, ATP-binding cassette transporters, elongation factor Tu, and SmpB were identified among the proteins associated with tmRNA of S. aureofaciens. We examined the functional role of ribosomal protein SS1 in a defined in vitro trans-translation system. Our data show that the protein SS1 that structurally differs from S1 of Escherichia coli is required for translation of the tmRNA tag-reading frame.
- MeSH
- bakteriální RNA genetika metabolismus MeSH
- buněčná diferenciace fyziologie MeSH
- financování organizované MeSH
- messenger RNA metabolismus MeSH
- molekulární sekvence - údaje MeSH
- proteiny vázající RNA metabolismus MeSH
- proteosyntéza MeSH
- retardační test MeSH
- ribonukleoproteiny metabolismus MeSH
- ribozomální proteiny metabolismus MeSH
- RNA transferová metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- spory bakteriální metabolismus MeSH
- Streptomyces coelicolor genetika růst a vývoj MeSH
