Most antibiotics are natural compounds or their derivatives, and bacteria have evolved defensive mechanisms to resist them. Many of these mechanisms are still poorly understood or unknown. This study reveals that in Bacillus subtilis, the transcription factor HelD increases resistance to rifampicin by protecting its target, RNA polymerase (RNAP). This protection is mediated by the HelD N-terminal domain that penetrates into RNAP to the close vicinity of the rifampicin binding pocket. Importantly, the bacterium detects low rifampicin levels using a unique regulatory system involving two convergent promoters with finely tuned kinetic properties. In the absence of rifampicin, the stronger antisense promoter inhibits transcription from the sense promoter. In the presence of subinhibitory rifampicin concentration, the antisense promoter is more likely to encounter rifampicin-bound RNAP. This relieves the repression from the sense promoter, increasing its transcription by almost two orders of magnitude, boosting helD expression. A similar two-promoter arrangement also controls the pps gene, which encodes a rifampicin-modifying enzyme. These findings define a widespread bacterial response system sensitive to rifampicin, as this dual-promoter architecture is conserved across many bacterial species and found upstream of genes potentially involved in rifampicin resistance, such as those for hydrolases, transporters, and transferases.

Introduction:The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars. Some PTS proteins share structural motifs with rifampicin phosphotransferases (RPHs), which inactivate rifampicin by phosphorylation. This homology suggests that the PTS may represent an evolutionary ancestor of the multi domain RPHs, though direct biochemical evidence has been lacking. Methods:Bacillus subtilis strains lacking genes encoding PTS proteins were evaluated in growth assays in the absence/presence of rifampicin; liquid chromatography-mass spectrometry was used to monitor the ability of B. subtilis PTS proteins to phosphorylate rifampicin; thermophoresis was employed to characterize protein-rifampicin interactions. Results:Deletion of B. subtilis ptsH, ptsI genes (encoding PTS proteins: HPr and EI) or rphT (encoding RphT-B. subtilis RPH) impaired growth in the presence of rifampicin. In vitro, the PTS complex (HPr, EI, MtlF, and PckA) phosphorylated rifampicin, with EI alone sufficient for this activity. However, no rifampicin phosphorylation by EI was detected in vivo. Heterologous expression of rphT then strongly increased rifampicin resistance, while ptsH/ptsI expression did not. Conclusion:This study shows that part of the PTS, protein EI, can phosphorylate rifampicin, supporting its evolutionary link to RPHs. We also establish that RphT, a putative rifampicin phosphotransferase misannotated as phosphoenolpyruvate synthase (Pps), is a bona fide rifampicin-modifying enzyme in B. subtilis. Finally, we demonstrate that derepressing RphT or its horizontal transfer confers high-level resistance to rifampicin.

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in the regulation of gene expression. We searched for CarD interaction partners in Mycobacterium smegmatis and identified a novel uncharacterized protein, named CrsL (MSMEG_5890). CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria, including pathogenic species such as Mycobacterium tuberculosis. CrsL interacts directly with CarD, adopting an ordered structure in the complex, and also binds RNAP, controlling CarD–RNAP association. ChIP-seq showed that CrsL associates with the promoters of actively transcribed genes and ∼75% of these regions are also associated with CarD. RNA-seq revealed ∼50% and ∼66% overlap in differentially expressed genes between CrsL and CarD knockdowns during the exponential and stationary phases, respectively. Among CrsL-regulated genes are DesA desaturase (MSMEG_5773) and DEAD/DEAH-box RNA helicase MSMEG_1930, which contribute to cold stress adaptation. CrsL supports the growth of M. smegmatis at elevated temperature but limits growth in cold environments. In summary, these findings identify CrsL as a novel, conserved CarD-interacting protein playing a key role in mycobacterial stress responses by modulating CarD function.

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. We searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two proteins: ApeB (MSMEG\_5828) and an uncharacterized protein, which we named CrsL (MSMEG\_5890). While ApeB interacted with CarD only when CarD was overexpressed, CrsL associated with CarD at its physiological levels. CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria including pathogenic species such as Mycobacterium tuberculosis. CrsL directly interacts with CarD and binds RNAP. ChIP-seq showed that CrsL associates with promoters of actively transcribed genes and ~75 % of these regions are also associated with CarD. RNA-seq showed ~50% and ~66% overlap in differentially expressed genes between CrsL and CarD knockdowns during exponential and stationary phases, respectively. CrsL represses expression of DesA desaturase (MSMEG\_5773) and DEAD/DEAH-box RNA helicase MSMEG\_1930, which are important for adaptation to cold stress. Furthermore, CrsL promotes the growth of M. smegmatis at elevated temperature. In summary, this study identifies CrsL as a novel actinobacterial transcription factor and provides a basis for its further investigation. ### Competing Interest Statement The authors have declared no competing interest.

HelD protein, also named HelR (encoded by MSMEG_2174 in Mycobacterium smegmatis), interacts with mycobacterial RNA polymerase (RNAP) and affects rifampicin resistance in Mycobacterium abscessus. Here, we provide data on rifampicin resistance and helD presence in the genomes of other clinically relevant nontuberculous mycobacteria. We show that helD is primarily found in rapidly growing mycobacteria, such as M. smegmatis, where we detected HelD at a subset of promoters that can also associate with CarD and RbpA. Transcriptome analysis of a helD deletion strain using RNA-seq revealed that HelD enhances gene expression during exponential growth and decreases it in stationary phase, during which we observed reduced levels of CarD, RbpA, and GTP, the initiation nucleotide for the majority of M. smegmatis transcripts. We propose a model in which HelD releases abortive RNAP complexes and confirm that HelD dissociates RNAP from the promoter in vitro. HelD not only helps mycobacteria overcome rifampicin treatment but also supports efficient transcription during rapid growth, which indicates a dual role of this transcription regulator.

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.

During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently co-localize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.

AbstractMycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

Abstract Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, are able to dissociate rifampicin-stalled RNAPs from DNA and provide rifampicin resistance. This is achieved by the ability of HelR proteins to dissociate rifampicin from RNAP. The HelR-mediated mechanism of rifampicin resistance is discussed here, and the roles of HelD/HelR in the transcriptional cycle are outlined. Moreover, the possibility that the structurally similar HelD proteins from low G+C Firmicutes may be also involved in rifampicin resistance is explored. Finally, the discovery of the involvement of HelR in rifampicin resistance provides a blueprint for analogous studies to reveal novel mechanisms of bacterial antibiotic resistance.

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 σ A 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 σ A and its shortened version lacking domain 1.1 ( σ A _ Δ 1.1 ). The results revealed that while full length σ A increases transcription activity of RNAP with increasing temperature, transcription with σ A _ Δ 1.1 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.

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.

: Five 2 '-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2 '-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.

The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analysis. This approach allowed us to identify the regulon of Bacillus subtilis sigma factor SigB of RNA polymerase (RNAP) specifically expressed during germination and outgrowth. SigB is critical for the cell’s response to general stress but is also expressed during spore germination and outgrowth, and this specific regulon is not known. This approach allowed us to (i) define a subset of the known SigB regulon controlled by SigB specifically during spore germination and outgrowth, (ii) identify the influence of the promoter sequence binding motif organization on the expression of the SigB-regulated genes, and (iii) suggest additional sigma factors co-controlling other SigB-dependent genes. Experiments then validated promoter sequence characteristics necessary for direct RNAP–SigB binding. In summary, this work documents the potential of computational approaches to unravel new information even for a well-studied system; moreover, the study specifically identifies the subset of the SigB regulon, which is activated during germination and outgrowth.

Bacterial RNA polymerase (RNAP) is an essential multisubunit enzyme performing transcription.Regulation of this process is secured through the stage-dependent interactions of RNAP with different factors (mostly proteins).Here we report the structurefunction analysis of the functional complexes between RNAP and a unique helicase-like factor HelD [1] which is present in many Gram-positive bacteria (e.g.Bacillus subtilis and Mycobacterium smegmatis) [2, 3].HelD forms tightly bound complexes with RNAP.It simultaneously penetrates into RNAP primary and secondary channels which are responsible for nucleic acids binding and substrate delivery.HelD can also interact with the RNAP active site.Structurally, these interactions are incompatible with the binding of DNA to the RNAP core and thus with the elongation stage of transcription.This is in accordance to our functional data showing that HelD is capable of clearing RNAP of nucleic acids and that HelD can dismantle RNAP-DNA complexes.HelD itself is composed of several domains, showing structural changes in solution [2] as well as in complexes with RNAP (three different structural states obtained from the cryo-EM analysis) [3].Although we were able to link the observed dynamic behaviour with the DNA-clearing role of HelD, the recycling of HelD-bound RNAP and subsequent restart of transcription remains to be explained.HelD as well as its complexes with RNAP resisted our attempts to crystallize them for many years.In order to get to the structural details we took the advantage of recent developments in the field of single-particle cryo-EM and were able to obtain ~3Å resolution structures.The structure of HelD itself was completely unknown with no homologue in the PDB.We combined X-ray crystallography (structure of one domain) and cryo-EM, together with bioinformatics and homologous modelling and successfully built de novo a complete atomic model of the HelD protein.For the analysis of condition-dependent dynamic behaviour we used small-angle X-ray scattering [2].Results from our structural studies were supplemented with biochemical and biophysical assays (enzymology, analysis of interactions and stability) and by computational analyses [3].