Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
- MeSH
- Bacillus subtilis genetika metabolismus MeSH
- bakteriální proteiny * metabolismus genetika MeSH
- bakteriální RNA * metabolismus genetika MeSH
- Corynebacterium glutamicum genetika metabolismus MeSH
- DNA řízené RNA-polymerasy * metabolismus genetika MeSH
- genetická transkripce MeSH
- konformace nukleové kyseliny MeSH
- Mycobacterium smegmatis genetika metabolismus enzymologie MeSH
- Mycobacterium tuberculosis genetika metabolismus MeSH
- nekódující RNA MeSH
- regulace genové exprese u bakterií MeSH
- sigma faktor * metabolismus genetika MeSH
- Streptomyces coelicolor genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Bordetella pertussis * metabolismus MeSH
- chromatografie kapalinová MeSH
- lidé MeSH
- pertuse * MeSH
- regulace genové exprese u bakterií MeSH
- sigma faktor genetika MeSH
- tandemová hmotnostní spektrometrie MeSH
- umělá inteligence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
σ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ factors occupies the primary channel of RNAP and also prevents binding of the σ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σA exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α helices, while the second α helix and β strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σA and its shortened version lacking domain 1.1 ( σA_Δ1.1 ). The results revealed that while full length σA increases transcription activity of RNAP with increasing temperature, transcription with σA_Δ1.1 remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.
- MeSH
- amidy metabolismus MeSH
- Bacillus subtilis * enzymologie MeSH
- DNA řízené RNA-polymerasy * chemie metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- protony MeSH
- rozbalení proteinů * MeSH
- sigma faktor * chemie metabolismus MeSH
- teplota * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.
A sigma (σ) factor is a constituent of bacterial RNA polymerase that guides the holoenzyme to promoter sequences and initiates transcription. In addition to a primary housekeeping σ factor, bacteria contain a number of alternative σ factors which recognize a specific set of promoters. By replacing the primary σ factor with alternative variants, the cell controls transcription of the whole sets of genes, typically to acclimate to changes in the environment. As key regulatory elements, σ factors are frequent targets of genetic manipulation aimed at the improvement of bacterial stress tolerance and capacity for bioproduction. Cyanobacteria are a phylum of bacteria capable of oxygenic photosynthesis and there is a great interest to employ them as biochemical and biofuel production hosts. Engineering of σ factor genes has become an important strategy to improve robustness and suitability of cyanobacteria for the production of high-value metabolites such as polyhydroxybutyrate, succinate, sucrose or hydrogen. Here, we summarize the current knowledge about the regulatory role of different σ factor classes in cyanobacteria, highlighting their biotechnological potential.
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
The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.
- MeSH
- Bacillus subtilis genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- genetická transkripce * MeSH
- operon MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u bakterií * MeSH
- regulon MeSH
- sigma faktor genetika MeSH
- transkriptom MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown. RESULTS: In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope. CONCLUSIONS: This study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.
This study describes the meta-analysis and kinetic modelling of gene expression control by sigma factor SigA of Bacillus subtilis during germination and outgrowth based on microarray data from 14 time points. The analysis computationally models the direct interaction among SigA, SigA-controlled sigma factor genes (sigM, sigH, sigD, sigX), and their target genes. Of the >800 known genes in the SigA regulon, as extracted from databases, 311 genes were analysed, and 190 were confirmed by the kinetic model as being controlled by SigA. For the remaining genes, alternative regulators satisfying kinetic constraints were suggested. The kinetic analysis suggested another 214 genes as potential SigA targets. The modelling was able to (i) create a particular SigA-controlled gene expression network that is active under the conditions for which the expression time series was obtained, and where SigA is the dominant regulator, (ii) suggest new potential SigA target genes, and (iii) find other possible regulators of a given gene or suggest a new mechanism of its control by identifying a matching profile of unknown regulator(s). Selected predicted regulatory interactions were experimentally tested, thus validating the model.
- MeSH
- Bacillus subtilis genetika MeSH
- bakteriální proteiny genetika MeSH
- genetická transkripce genetika MeSH
- genové regulační sítě genetika MeSH
- kinetika MeSH
- regulace genové exprese u bakterií genetika MeSH
- sigma faktor genetika MeSH
- spory bakteriální genetika MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- metaanalýza MeSH
- práce podpořená grantem MeSH
Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.
- MeSH
- Bacillus subtilis metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- DNA bakterií chemie metabolismus MeSH
- DNA řízené RNA-polymerasy chemie genetika metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- izotopy dusíku MeSH
- izotopy uhlíku MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- konzervovaná sekvence MeSH
- molekulární modely * MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- podjednotky proteinů MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- sigma faktor chemie genetika metabolismus MeSH
- stabilita proteinů MeSH
- strukturní homologie proteinů MeSH
- Thermotoga maritima enzymologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
