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.

• Klíčová slova
• Corynebacterium glutamicum, Mycomembrane, SigD, Sigma factor, Trehalose dicorynomycolate,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Corynebacterium glutamicum genetika   růst a vývoj   metabolismus   MeSH
• kyseliny mykolové metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• sigma faktor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• corynomycolic acid MeSH Prohlížeč
• kyseliny mykolové MeSH
• sigma faktor MeSH

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences, and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high levels and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that led to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally encoded transcriptional repressor protein AbrB, thereby derepressing a potent antisense transcript that antagonized SigN expression. SigN efficiently competed with the vegetative sigma factor SigA in vitro, and SigN accumulation in the absence of positive feedback reduced SigA-dependent transcription suggesting that toxicity may be due to competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a toxic sigma factor is unclear but SigN may function in host-inhibition during lytic conversion, as phage lysogen genes are also encoded on pBS32. IMPORTANCE Alternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded alternative sigma factor SigN of Bacillus subtilis however, is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

• Klíčová slova
• AbrB, SigN, cell death, pBS32, plasmid, prophage,
• MeSH
• Bacillus subtilis * genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• genetická transkripce MeSH
• imunoglobulin A sekreční genetika   MeSH
• regulace genové exprese u bakterií MeSH
• sigma faktor * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• imunoglobulin A sekreční MeSH
• sigma faktor * MeSH

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

• Klíčová slova
• Rhodococcus erythropolis, in vitro transcription, RNA-seq, sigma factor, transcriptional regulatory network,
• MeSH
• bakteriální proteiny * metabolismus   MeSH
• genetická transkripce MeSH
• genové regulační sítě MeSH
• regulace genové exprese u bakterií * MeSH
• Rhodococcus MeSH
• sigma faktor metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• sigma faktor 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.

• Klíčová slova
• Bordetella pertussis, CesT chaperone, T3SS, anti-sigma factor, biofilm,
• 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
• Názvy látek
• bakteriální proteiny MeSH
• sigma faktor MeSH

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.

• Klíčová slova
• Anabaena, Biofuel, Cyanobacteria, Environmental stress, Metabolites, RNA polymerase, Sigma factor, Synechocystis,
• MeSH
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• fotosyntéza MeSH
• genetická transkripce MeSH
• sigma faktor MeSH
• sinice * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• sigma faktor MeSH

Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• DNA footprinting MeSH
• Escherichia coli genetika   MeSH
• genetická transkripce * MeSH
• geny rRNA MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• plazmidy MeSH
• promotorové oblasti (genetika) MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• sekvenční seřazení MeSH
• sigma faktor chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Streptomyces coelicolor fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• sigma faktor MeSH

The Corynebacterium glutamicum genome codes for 7 sigma subunits (factors) of RNA polymerase (RNAP): primary sigma factor SigA (σ(A)), primary-like SigB and 5 other alternative sigma factors (SigC, SigD, SigE, SigH and SigM). Each sigma factor is responsible for recognizing promoters of genes belonging to a regulon (sigmulon) involved in specific functions of the cell. Most promoters of C. glutamicum housekeeping genes are recognized by RNAP+σ(A), whereas σ(B) is involved in transcription of a large group of genes active during the transition phase between the exponential and stationary growth phases when various stress factors threaten to damage the cell. The σ(H) regulon consists of the genes involved in heat shock response including those coding for regulators and other sigma factors. It seems therefore that σ(H) occupies a central position in the cross-regulated network of sigma factors and controls their concerted response to various stress conditions in C. glutamicum. The σ(M) factor was found to regulate genes responding to oxidative stress. The main role of σ(E) is to activate genes involved in response to a cell surface stress. Promoters of individual classes recognized by different sigma factors are compiled and the respective consensus sequences of their key recognition motifs (-35 and -10 regions) are derived. In a number of genes, two or more promoters controlled by the same or different sigma factors were discovered. These multiple, overlapping or dual promoters contribute to a complex gene transcription control mechanisms that integrate internal and external signals and tune gene expression in cells as required by environmental and physiological conditions.

• MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• fyziologický stres genetika   MeSH
• genové regulační sítě MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií * MeSH
• regulon MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sigma faktor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• sigma faktor MeSH

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• genetická transkripce * MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií * MeSH
• sigma faktor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• sigma faktor MeSH

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
• Názvy látek
• sigma faktor 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.

• MeSH
• Corynebacterium glutamicum genetika   MeSH
• DNA řízené RNA-polymerasy * genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• Rhodococcus * enzymologie   genetika   MeSH
• sigma faktor * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA řízené RNA-polymerasy * MeSH
• sigma faktor * MeSH