Добірка наукової літератури з теми "(p)ppGpp Synthetases"

The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. InBacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that inB. subtilis, arelAmutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that therelAmutant is trapped in a SigD “on” state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility inB. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5′-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.

Suppression of the stringent response is a promising strategy for the treatment of persistent bacterial infections. A novel class of compounds having a mechanism of action based on alarmone synthetase inhibition and suppressing the synthesis of (p)ppGpp alarmones in bacteria may provide a more effective treatment for latent infections and resolve problems associated with bacterial persistence. Conventional antibiotics primarily act on actively growing bacteria, but they are inactive against persister cells with a slowed metabolism. Alarmone synthetase inhibitors have antipersister properties that may enhance conventional antibiotics’ antibacterial action. Two groups of RSH proteins are responsible for the synthesis of alarmones: long RelA/SpoT homologs and small alarmone synthetases. Many species of bacteria possess both types of enzymes. Despite the fact that a number of inhibitors of bifunctional long synthetases/hydrolases have been described to date, their properties with respect to monofunctional small alarmone synthetases have been studied poorly. This study investigated the effect of the alarmone synthetase inhibitor DMNP on the purified RelZ small alarmone synthetase protein from Mycolicibacterium smegmatis.

ABSTRACT The RelA, RelP, and RelQ enzymes are responsible for the production of the alarmone (p)ppGpp in Streptococcus mutans. A strain lacking all three synthetases (ΔrelAPQ) does not grow in minimal medium lacking the branched-chain amino acids (BCAA) leucine or valine but grows well if isoleucine is also omitted. Here, we investigated whether there was a correlation between growth in the absence of leucine and valine with (p)ppGpp pools and the activation of CodY. By using a combination of single, double, and triple mutants lacking the (p)ppGpp synthetase enzymes, we demonstrated that the ability to grow in the absence of leucine or valine required basal levels of (p)ppGpp production by RelP and RelQ. The introduction of a codY mutation into the ΔrelAPQ strain fully restored growth in medium lacking leucine or valine, revealing that the growth-defective phenotype of ΔrelAPQ was directly linked to CodY. Lowering GTP levels through the addition of decoyinine did not alleviate CodY repression or affect the expression of genes involved in BCAA biosynthesis, suggesting that S. mutans CodY is not activated by GTP. The results of phenotypic studies revealed that the codY mutant had a reduced capacity to form biofilms and that its growth was more sensitive to low pH, showing a role for CodY in two key virulence properties of S. mutans. Microarray results revealed the extent of the CodY regulon. Notably, the identification of putative CodY-binding boxes upstream of genes that were downregulated in the codY mutant indicates that CodY may also function as a transcriptional activator in S. mutans.

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin−antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS–antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

ABSTRACT In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

Adaptation to a rapidly fluctuating environment is the key to the survival of an organism. Bacteria sense and respond to stress by an overall reprogramming of the cellular processes to shut down the energy-consuming processes and switch to pathways that ensure the survival under stress. One of the strategies utilized by bacteria is to mount ‘stringent response’ which is mediated by the second messengers (p)ppGpp. (p)ppGpp governs a multitude of phenotypes in Mycobacteria and for a long time the bifunctional Rel was believed to be its only (p)ppGpp synthetase. A serendipitous detection of (p)ppGpp in a Mycobacterium smegmatis strain devoid of Rel led to the discovery of a second (p)ppGpp synthetase thereby broadening the horizon of stringent response in Mycobacteria (Murdeshwar and Chatterji, 2012). This unique protein contained a RNaseH domain along with the (p)ppGpp synthesis domain suggesting a role distinct from that of Rel. Subsequent characterisation of the protein revealed that neither domain is active in isolation raising a question about the link between these activities. Due to the crucial role played by (p)ppGpp, it becomes essential to analyse the (p)ppGpp null phenotype. Several bacterial species like Bacillus subtilis have short alarm one synthetases in addition to Rel and they have been proposed to be activated under particular stress conditions underlining the need to delineate the role of these (p)ppGpp synthetases (Nanamiya et al., 2008). Our study proposes a role for MS_RHII-RSD in vivo and deals with the phenotypic characterisation of the Δrel Δms_rhII-rsd strain. Chapter 1 reviews the available literature in the field of stringent response and provides the rationale behind this study. The discovery of (p)ppGpp and the plethora of functions regulated by it is explained along with a description of the key players in the (p)ppGpp metabolism. The chapter stresses upon the need to investigate the significance of a second (p)ppGpp synthetase in Mycobacteria and the scope of the current study. Chapter 2 deals with the elucidation of the in vivo significance of MS_RHII-RSD in M. smegmatis and proposes a role for the protein in R-loop removal during stress which requires both RNaseH activity and (p)ppGpp synthesis. The in vitro R-loop hydrolysis assays along with evidence for R-loop removal in M. smegmatis have been discussed along with the strategy used for the generation of the Δms_rhII-rsd strain. Chapter 3 explores the interdependence between the RNaseH and (p)ppGpp domains in MS_RHII-RSD in an attempt to unravel the necessity of the RNase H activity in a (p)ppGpp synthetase. The generation of active-site mutants of RNaseH and RSD along with their functional and biophysical characterisation has been described in detail. Oligomerisation studies with MS_RHII-RSD revealed the importance of a hexameric form for the protein. Chapter 4 further elaborates upon the link between the RNaseH activity and the (p)ppGpp synthesis activity and reveals a possible regulation of (p)ppGpp synthesis activity by RNA. Furthermore, the differing substrate specificities between Rel and MS_RHII-RSD are discussed. A possibility of the presence of pGpp due to MS_RHII-RSD in Mycobacteria has been outlined. Chapter 5 describes the attempts at generating a (p)ppGpp-deficient strain of M. smegmatis and reveals the surprising presence of yet another (p)ppGpp synthetase. The generation and characterisation of the Δrel Δms_rhII-rsd strain was performed and the physiological role of MS_RHII-RSD in biofilm formation and antibiotic tolerance has been highlighted. Chapter 6 summarizes the results of the study and points out the future directions for the work. Appendix 1 gives a comprehensive list of strains and plasmids used in this study. Appendix 2 provides a list of growth differences in antibiotics between the wild type and knockout strains of M. smegmatis obtained by Phenotype microarray. Appendix 3 is a commentary on the Pup-proteasome regulation in Mycobacteria.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.