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
HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- bakteriální RNA genetika MeSH
- chromatinová imunoprecipitace MeSH
- DNA bakterií chemie metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- exprese genu MeSH
- geny rRNA MeSH
- kinetika MeSH
- modely genetické MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- regulon * MeSH
- RNA transferová genetika MeSH
- sekvenční analýza DNA MeSH
- sigma faktor metabolismus MeSH
- Streptomyces coelicolor genetika metabolismus MeSH
- vazebná místa 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.
A computational model of gene expression was applied to a novel test set of microarray time series measurements to reveal regulatory interactions between transcriptional regulators represented by 45 sigma factors and the genes expressed during germination of a prokaryote Streptomyces coelicolor. Using microarrays, the first 5.5 h of the process was recorded in 13 time points, which provided a database of gene expression time series on genome-wide scale. The computational modeling of the kinetic relations between the sigma factors, individual genes and genes clustered according to the similarity of their expression kinetics identified kinetically plausible sigma factor-controlled networks. Using genome sequence annotations, functional groups of genes that were predominantly controlled by specific sigma factors were identified. Using external binding data complementing the modeling approach, specific genes involved in the control of the studied process were identified and their function suggested.
- MeSH
- genetická transkripce MeSH
- genové regulační sítě * MeSH
- kinetika MeSH
- modely genetické * MeSH
- počítačová simulace MeSH
- regulace genové exprese u bakterií * MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- sigma faktor metabolismus MeSH
- spory bakteriální genetika růst a vývoj metabolismus MeSH
- stanovení celkové genové exprese * MeSH
- Streptomyces coelicolor genetika metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Streptomycetes have been studied mostly as producers of secondary metabolites, while the transition from dormant spores to an exponentially growing culture has largely been ignored. Here, we focus on a comparative analysis of fluorescently and radioactively labeled proteome and microarray acquired transcriptome expressed during the germination of Streptomyces coelicolor. The time-dynamics is considered, starting from dormant spores through 5.5 hours of growth with 13 time points. Time series of the gene expressions were analyzed using correlation, principal components analysis and an analysis of coding genes utilization. Principal component analysis was used to identify principal kinetic trends in gene expression and the corresponding genes driving S. coelicolor germination. In contrast with the correlation analysis, global trends in the gene/protein expression reflected by the first principal components showed that the prominent patterns in both the protein and the mRNA domains are surprisingly well correlated. Analysis of the number of expressed genes identified functional groups activated during different time intervals of the germination.
- MeSH
- analýza hlavních komponent MeSH
- energetický metabolismus genetika MeSH
- fenotyp MeSH
- fyziologický stres genetika MeSH
- genové regulační sítě MeSH
- metabolické sítě a dráhy MeSH
- proteom * MeSH
- regulace genové exprese u bakterií * MeSH
- spory bakteriální genetika metabolismus MeSH
- Streptomyces coelicolor genetika metabolismus ultrastruktura MeSH
- transkriptom * 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 occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.
- MeSH
- aminoacylace tRNA MeSH
- fosforylace MeSH
- peptidyltransferasy metabolismus MeSH
- podjednotky ribozomu metabolismus MeSH
- poly U genetika metabolismus MeSH
- ribozomální proteiny metabolismus MeSH
- Streptomyces coelicolor enzymologie genetika metabolismus MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
