Mycobacterial 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.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases * metabolism   MeSH
• Transcription, Genetic MeSH
• Transcription Initiation, Genetic * MeSH
• Mycobacterium smegmatis * metabolism   genetics   MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Rifampin * pharmacology   MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Transcription Factors metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Bacterial Proteins * MeSH
• DNA-Directed RNA Polymerases * MeSH
• Rifampin * MeSH
• Sigma Factor * MeSH
• Transcription Factors MeSH

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.

• MeSH
• Bacillus subtilis * genetics   metabolism   MeSH
• Bacterial Proteins * chemistry   metabolism   genetics   MeSH
• DNA, Bacterial metabolism   chemistry   genetics   MeSH
• DNA-Binding Proteins chemistry   metabolism   genetics   MeSH
• DNA chemistry   metabolism   MeSH
• Cryoelectron Microscopy * MeSH
• Fructosediphosphates MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular * MeSH
• Protein Multimerization MeSH
• Operon genetics   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Repressor Proteins * chemistry   metabolism   genetics   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• DNA, Bacterial MeSH
• DNA-Binding Proteins MeSH
• DNA MeSH
• fructose-1,6-diphosphate MeSH Browser
• Fructosediphosphates MeSH
• Repressor Proteins * MeSH

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, β, β'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

• MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Genes, Bacterial MeSH
• Gene Deletion MeSH
• DNA-Directed RNA Polymerases chemistry   genetics   metabolism   MeSH
• Gene Duplication MeSH
• Endoribonucleases genetics   physiology   MeSH
• Transcription, Genetic MeSH
• Homeostasis MeSH
• RNA, Messenger metabolism   MeSH
• Evolution, Molecular MeSH
• Mutation MeSH
• Suppression, Genetic MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Endoribonucleases MeSH
• RNA, Messenger MeSH

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.

• Keywords
• Bacillus subtilis, DNA topology, ribosomal RNA, transcription,
• Publication type
• Journal Article MeSH

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Stress, Physiological * MeSH
• Ligases genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Staphylococcus aureus genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Browser
• Ligases MeSH

Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.

• MeSH
• Bacillus subtilis genetics   MeSH
• Bacterial Proteins genetics   MeSH
• Ligases genetics   MeSH
• Heat-Shock Response genetics   MeSH
• Gene Expression Regulation, Bacterial genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Browser
• Ligases MeSH

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP.IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

• Keywords
• RNA polymerase, bacterial transcription, conformational change, cryo-electron microscopy, mycobacteria, protein structure, transcription initiation factor,
• MeSH
• DNA-Directed RNA Polymerases chemistry   ultrastructure   MeSH
• Cryoelectron Microscopy MeSH
• Holoenzymes chemistry   ultrastructure   MeSH
• Protein Conformation MeSH
• Mycobacterium smegmatis enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Holoenzymes MeSH

For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these 2 isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80S initiation complex.

• Keywords
• ABCE1, cryo-EM, recycling, ribosome, translation,
• MeSH
• ATP-Binding Cassette Transporters chemistry   metabolism   MeSH
• Peptide Elongation Factors MeSH
• Hydrolysis MeSH
• Peptide Chain Initiation, Translational * MeSH
• Peptide Initiation Factors metabolism   MeSH
• Rabbits MeSH
• Models, Molecular MeSH
• Nucleosides chemistry   MeSH
• Ribosomes metabolism   MeSH
• Peptide Chain Termination, Translational MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Peptide Elongation Factors MeSH
• Peptide Initiation Factors MeSH
• Nucleosides MeSH

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

• MeSH
• Bacillus subtilis enzymology   MeSH
• Deoxyribonucleotides biosynthesis   chemistry   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Escherichia coli enzymology   MeSH
• Transcription, Genetic * MeSH
• Templates, Genetic MeSH
• Nucleic Acid Conformation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deoxyribonucleotides MeSH
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH

The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity towards Gram-positive bacteria that include pathogens and resistant strains. For further development of these compounds, it was necessary to identify the mechanism of their action and characterize their interaction with eukaryotic cells/organisms in more detail. Here, we show that at their bactericidal concentrations LPPOs localize to the plasmatic membrane in bacteria but not in eukaryotes. In an in vitro system we demonstrate that LPPOs create pores in the membrane. This provides an explanation of their action in vivo where they cause serious damage of the cellular membrane, efflux of the cytosol, and cell disintegration. Further, we show that (i) LPPOs are not genotoxic as determined by the Ames test, (ii) do not cross a monolayer of Caco-2 cells, suggesting they are unable of transepithelial transport, (iii) are well tolerated by living mice when administered orally but not peritoneally, and (iv) are stable at low pH, indicating they could survive the acidic environment in the stomach. Finally, using one of the most potent LPPOs, we attempted and failed to select resistant strains against this compound while we were able to readily select resistant strains against a known antibiotic, rifampicin. In summary, LPPOs represent a new class of compounds with a potential for development as antibacterial agents for topical applications and perhaps also for treatment of gastrointestinal infections.

• MeSH
• Biological Transport, Active MeSH
• Anti-Bacterial Agents chemistry   pharmacokinetics   pharmacology   MeSH
• Bacillus subtilis drug effects   growth & development   metabolism   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Caco-2 Cells MeSH
• Enterococcus faecalis drug effects   growth & development   MeSH
• Gram-Positive Bacteria drug effects   metabolism   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Molecular Structure MeSH
• Mice, Inbred ICR MeSH
• Mice MeSH
• Drug Discovery MeSH
• Cell Membrane Permeability drug effects   MeSH
• Pyrimidine Nucleosides chemistry   pharmacokinetics   pharmacology   MeSH
• Drug Stability MeSH
• Streptococcus agalactiae drug effects   growth & development   MeSH
• Microscopy, Electron, Transmission MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Pyrimidine Nucleosides MeSH