The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx–RNA polymerase (Spx–RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx–RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx–RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended −35 box in which nucleotide composition at the −43/−44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

dRNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the ␦ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of ␦ from Bacillus subtilis. We demonstrate that ␦ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that ␦ is essential for cell survival when facing a competing strain in a changing environment. Hence, although ␦ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis. RNA polymerase (RNAP) is the key enzyme responsible for transcription of DNA into RNA. Bacterial RNAP core enzyme consists of several subunits: the ␣ dimer that holds together ␤ and ␤=, which form the catalytic center, and the subunit that binds to ␤=. This core enzyme, ␣ 2 ␤␤=, is capable of elongating but not initiating transcription. To initiate transcription, the core enzyme must associate with a subunit that allows the holoenzyme to recognize specific sequences in the DNA, i.e., promoters. Typically, several different subunits are present in the cell and direct the expression of different subsets of genes (1, 2).While the ␣ 2 ␤␤= composition is conserved across the bacterial kingdom, Gram-positive Firmicutes contain an additional subunit, ␦, which is encoded by the rpoE gene in the model bacterium Bacillus subtilis. The ␦ subunit was first reported as an endogenous protein present in RNAP from phage SP01-infected Bacillus subtilis cells, which was required for its accurate middle gene transcription (3, 4). The rpoE gene specifies a protein of 173 amino acids (aa) with a molecular mass of ϳ20.5 kDa. The protein is highly acidic (pI, 3.6) (5). As determined by circular dichroism (CD) spectroscopy, it consists of two domains: (i) the N-terminal domain (NTD), which is structured; and (ii) the C-terminal domain, which is unstructured and whose amino acid composition-stretches of glutamic and aspartic acid residues-makes it virtually a polyanion (6). The structure of the ordered N-terminal domain was recently solved based on a truncated construct consisting of the N-terminal domain containing a His tag. The NTD contains four ␣-helices and an antiparallel ␤-sheet (7). Delta binds to RNAP in vivo (8), but the binding site is unknown.The in vitro effects of ␦ on transcription were previously examined in detail in the B. subtilis system. ␦ was reported to destabilize complexes between RNAP and DNA in vitro, thus increasing RNAP's specificity for good consensus promoter sequences (9, 10). Despite this inhibitory effect, ␦ was shown to stimulate transcription on some temp...

Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i-iii. Lipophosphonoxins displayed significant antibacterial properties against a panel of Gram-positive species, including multiresistant strains. The minimum inhibitory concentration (MIC) values of the best inhibitors were in the 1-12 μg/mL range, while their cytotoxic concentrations against human cell lines were significantly above this range. The modular nature of this artificial scaffold offers a large number of possibilities for further modifications/exploitation of these compounds.

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well-folded N-terminal domain was determined based on inter-proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C-terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C-terminal domain. Various (15) N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C-terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C-terminal tail with the N-terminal domain and with itself were identified. A propensity of the C-terminal domain to form β-type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well-balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C-terminal domain. The results showed that the δ subunit consists of a well-ordered N-terminal domain and a flexible C-terminal domain that exhibits a complex hierarchy of partial ordering.

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 in complex with RNAP and from 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 We found that 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 σ1.1. Moreover, modeling studies suggested that σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas σ1.1 must be rearranged to fit therein. Thus, the mesophilic species and share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in , 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.