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.

• Klíčová slova
• Bacillus subtilis, DNA topology, ribosomal RNA, transcription,
• Publikační typ
• časopisecké články 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
• bakteriální proteiny genetika   metabolismus   MeSH
• fyziologický stres * MeSH
• ligasy genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Staphylococcus aureus genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Prohlížeč
• ligasy 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 genetika   MeSH
• bakteriální proteiny genetika   MeSH
• ligasy genetika   MeSH
• reakce na tepelný šok genetika   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Prohlížeč
• ligasy MeSH

The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli We applied DRaCALA (differential radial capillary action of ligand assay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets in E. coli that, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp in E. coli are identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved between E. coli and Firmicutes In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets in E. coliIMPORTANCE Antibiotic resistance and tolerance exhibited by pathogenic bacteria have resulted in a global public health crisis. Remarkably, almost all bacterial pathogens require the alarmone (p)ppGpp to be virulent. Thus, (p)ppGpp not only induces tolerance of nutritional limitations and chemical insults, including antibiotics, but is also often required for induction of virulence genes. However, understanding of the molecular targets of (p)ppGpp and the mechanisms by which (p)ppGpp influences bacterial physiology is incomplete. In this study, a systematic approach was used to uncover novel targets of (p)ppGpp in E. coli, the best-studied model bacterium. Comprehensive comparative studies of the targets revealed conserved target pathways of (p)ppGpp in both Gram-positive and -negative bacteria and novel targets of (p)ppGpp, including an alternative degradative pathway of (p)ppGpp. Thus, our discoveries may help in understanding of how (p)ppGpp increases the stress resilience and multidrug tolerance not only of the model organism E. coli but also of the pathogenic organisms in which these targets are conserved.

• Klíčová slova
• (p)ppGpp, DRaCALA, GTPase, Nudix, multidrug tolerance, persistence,
• MeSH
• Escherichia coli metabolismus   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guanosinpentafosfát MeSH
• guanosintetrafosfát MeSH
• proteiny z Escherichia coli MeSH

The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

• Klíčová slova
• RelA, antibiotics, mupirocin, persistence, ppGpp, ribosomes, thiostrepton, tolerance, trimethoprim, β-lactam,
• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamy farmakologie   MeSH
• chloramfenikol farmakologie   MeSH
• dihydrofolátreduktasa genetika   metabolismus   MeSH
• Escherichia coli chemie   genetika   metabolismus   MeSH
• guanosintetrafosfát analogy a deriváty   metabolismus   MeSH
• isoleucin-tRNA-ligasa genetika   MeSH
• lékové interakce MeSH
• ligasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• mupirocin farmakologie   MeSH
• proteosyntéza účinky léků   MeSH
• ribozomy účinky léků   metabolismus   MeSH
• RNA transferová genetika   metabolismus   MeSH
• subcelulární frakce chemie   účinky léků   metabolismus   MeSH
• tetracyklin farmakologie   MeSH
• thiostrepton farmakologie   MeSH
• tolerance léku * MeSH
• trimethoprim farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosine 5',5'''-triphosphoguanosine-3'''-diphosphate MeSH Prohlížeč
• antibakteriální látky MeSH
• beta-laktamy MeSH
• chloramfenikol MeSH
• dihydrofolátreduktasa MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Prohlížeč
• guanosintetrafosfát MeSH
• isoleucin-tRNA-ligasa MeSH
• ligasy MeSH
• mupirocin MeSH
• RNA transferová MeSH
• tetracyklin MeSH
• thiostrepton MeSH
• trimethoprim MeSH

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• deoxyguanosin analogy a deriváty   biosyntéza   chemie   MeSH
• dipeptidy biosyntéza   chemie   MeSH
• Enterococcus faecalis účinky léků   enzymologie   genetika   metabolismus   MeSH
• fyziologický stres MeSH
• guanosindifosfát metabolismus   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát biosyntéza   MeSH
• guanosintrifosfát metabolismus   MeSH
• hořčík MeSH
• ligasy genetika   metabolismus   MeSH
• molekulární struktura MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny MeSH
• deoxyguanosin MeSH
• dipeptidy MeSH
• guanosindifosfát MeSH
• guanosinpentafosfát MeSH
• guanosintetrafosfát MeSH
• guanosintrifosfát MeSH
• hořčík MeSH
• ligasy MeSH
• relacin MeSH Prohlížeč

RNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the δ 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 δ from Bacillus subtilis. We demonstrate that δ 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 δ is essential for cell survival when facing a competing strain in a changing environment. Hence, although δ 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.

• MeSH
• Bacillus subtilis enzymologie   genetika   fyziologie   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• exprese genu MeSH
• fyziologická adaptace MeSH
• genetická transkripce MeSH
• genový knockout MeSH
• mikrobiální viabilita MeSH
• podjednotky proteinů MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• podjednotky proteinů MeSH