In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

• Klíčová slova
• MoaB2, RNA polymerase, mycobacteria, transcription, σA,
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• genetická transkripce MeSH
• krystalografie rentgenová MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory * metabolismus   genetika   MeSH
• vazba proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy MeSH
• sigma faktor * MeSH
• transkripční faktory * MeSH

Bacillus subtilis is a model organism used to study molecular processes in Gram-positive bacteria. Sigma factor B, which associates with RNA polymerase, is one of the transcriptional regulators involved in the cell's response to environmental stress. Experiments have proven that the amounts of free σB (SigB) are controlled by a system of anti- (RsbW) and anti-anti-sigma (RsbV) factors expressed from the same operon as SigB. Moreover, the phosphorylation state of RsbV is controlled by phosphatases RsbP and RsbU, which directly dephosphorylate RsbV. A set of chemical equations describing the network controlling the levels of free SigB was converted to a set of differential equations quantifying the dynamics of the network. The solution of these equations allowed the simulation of the kinetic behavior of the network and its components under real conditions reflected in the time series of protein expression. In this study, the time series of protein expression measured by mass spectrometry were utilized to investigate the role of phosphatases RsbU/RsbP in transmitting the environmental signal. Additionally, the influence of kinetic constants and the amounts of other network components on the functioning of the network was investigated. A comparison with the same simulation performed using a transcriptomic dataset showed that while the time series between the proteomic and transcriptomic datasets are not correlated, the results are the same. This indicates that when modeling is performed within one dataset, it does not matter whether the data come from the mRNA or protein level. In summary, the computational results based on experimental data provide a quantitative insight into the functioning of the SigB-dependent circuit and offer a template for the quantitative study of similar systems.

• Klíčová slova
• Bacillus subtilis, computational modeling, protein regulatory networks, sigma B,
• Publikační typ
• časopisecké články MeSH

σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σE factor from Streptomyces coelicolor. The direct activator/repressor function of σE was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σE interactome was defined. Taken together, the results characterize σE, its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms.

• MeSH
• počítačová simulace MeSH
• sigma faktor genetika   MeSH
• Streptomyces coelicolor * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 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

Bacillus subtilis is a model organism used to study molecular processes in prokaryotic cells. Sigma factor B, which associates with RNA polymerase, is one of the transcriptional regulators involved in the cell's response to environmental stress. This study addresses the key question of how the levels of free SigB, which acts as the actual regulator of gene expression, are controlled. A set of chemical equations describing the network controlling the levels of free SigB was designed, leading to a set of differential equations quantifying the dynamics of the network. Utilizing a microarray-measured gene expression time series then allowed the simulation of the kinetic behavior of the network in real conditions and investigation of the role of phosphatases RsbU/RsbP transmitting the environmental signal and controlling the amounts of free SigB. Moreover, the role of kinetic constants controlling the formation of the molecular complexes, which consequently influence the amount of free SigB, was investigated. The simulation showed that although the total amount of sigma B is relatively high in the unstressed population, the amount of free SigB, which actually controls its regulon, is quite low. The simulation also allowed determination of the proportion of all the network members that were free or bound in complexes. While previously the qualitative features of B. subtilis SigB have been studied in detail, the kinetics of the network have mostly been ignored. In summary, the computational results based on experimental data provide a quantitative insight into the functioning of the SigB-dependent circuit and provide a roadmap for its further exploration in this industrially important bacterium.

• Klíčová slova
• Bacillus subtilis sigma B, computer simulation, regulatory network,
• Publikační typ
• časopisecké články MeSH