SMART MODE:

NORMAL GENOMIC

• de Almeida LG, Ortiz JH, Schneider RP, Spira B
• phoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate.
• Appl Environ Microbiol. 2015; 81: 3006-15
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• Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation.

• Germain E, Roghanian M, Gerdes K, Maisonneuve E
• Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases.
• Proc Natl Acad Sci U S A. 2015; 112: 5171-6
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• The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.

• de la Fuente-Nunez C, Reffuveille F, Haney EF, Straus SK, Hancock RE
• Broad-spectrum anti-biofilm peptide that targets a cellular stress response.
• PLoS Pathog. 2014; 10: 1004152-1004152
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• Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

• Spira B, Ferreira GM, de Almeida LG
• relA enhances the adherence of enteropathogenic Escherichia coli.
• PLoS One. 2014; 9: 91703-91703
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• Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele.

• Gopalkrishnan S, Nicoloff H, Ades SE
• Co-ordinated regulation of the extracytoplasmic stress factor, sigmaE, with other Escherichia coli sigma factors by (p)ppGpp and DksA may be achieved by specific regulation of individual holoenzymes.
• Mol Microbiol. 2014; 93: 479-93
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• The E. coli alternative sigma factor, sigma(E) , transcribes genes required to maintain the cell envelope and is activated by conditions that destabilize the envelope. sigma(E) is also activated during entry into stationary phase in the absence of envelope stress by the alarmone (p)ppGpp. (p)ppGpp controls a large regulatory network, reducing expression of sigma(70) -dependent genes required for rapid growth and activating sigma(70) -dependent and alternative sigma factor-dependent genes required for stress survival. The DksA protein often potentiates the effects of (p)ppGpp. Here we examine regulation of sigma(E) by (p)ppGpp and DksA following starvation for nutrients. We find that (p)ppGpp is required for increased sigma(E) activity under all conditions tested, but the requirement for DksA varies. DksA is required during amino acid starvation, but is dispensable during phosphate starvation. In contrast, regulation of sigma(S) is (p)ppGpp- and DksA-dependent under all conditions tested, while negative regulation of sigma(70) is DksA- but not (p)ppGpp-dependent during phosphate starvation, yet requires both factors during amino acid starvation. These findings suggest that the mechanism of transcriptional regulation by (p)ppGpp and/or DksA cannot yet be explained by a unifying model and is specific to individual promoters, individual holoenzymes, and specific starvation conditions.

• Nowicki D et al.
• Phenethyl isothiocyanate inhibits shiga toxin production in enterohemorrhagic Escherichia coli by stringent response induction.
• Antimicrob Agents Chemother. 2014; 58: 2304-15
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• The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) depends on production of Shiga toxins, which are encoded by stx genes located in the genomes of lambdoid prophages. Efficient expression of these genes requires prophage induction and lytic development of phages. Treatment of EHEC infections is problematic due to not only the resistance of various strains to antibiotics but also the fact that many antibiotics cause prophage induction, thus resulting in high-level expression of stx genes. Here we report that E. coli growth, Shiga toxin-converting phage development, and production of the toxin by EHEC are strongly inhibited by phenethyl isothiocyanate (PEITC). We demonstrate that PEITC induces the stringent response in E. coli that is mediated by massive production of a global regulator, guanosine tetraphosphate (ppGpp). The stringent response induction arises most probably from interactions of PEITC with amino acids and from amino acid deprivation-mediated activation of ppGpp synthesis. In mutants unable to synthesize ppGpp, development of Shiga toxin-converting phages and production of Shiga toxin are significantly enhanced. Therefore, ppGpp, which appears at high levels in bacterial cells after stimulation of its production by PEITC, is a negative regulator of EHEC virulence and at the same time efficiently inhibits bacterial growth. This is in contrast to stimulation of virulence of different bacteria by this nucleotide reported previously by others.

• Geiger T, Kastle B, Gratani FL, Goerke C, Wolz C
• Two small (p)ppGpp synthases in Staphylococcus aureus mediate tolerance against cell envelope stress conditions.
• J Bacteriol. 2014; 196: 894-902
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• The stringent response is a conserved global regulatory mechanism that is related to the synthesis of (p)ppGpp nucleotides. Gram-positive bacteria, such as Staphylococcus aureus, possess three (p)ppGpp synthases: the bifunctional RSH (RelA/SpoT homolog) protein, which consists of a (p)ppGpp synthase and a (p)ppGpp hydrolase domain, and two truncated (p)ppGpp synthases, designated RelP and RelQ. Here, we characterized these two small (p)ppGpp synthases. Biochemical analyses of purified proteins and in vivo studies revealed a stronger synthetic activity for RelP than for RelQ. However, both enzymes prefer GDP over GTP as the pyrophosphate recipient to synthesize ppGpp. Each of the enzymes was shown to be responsible for the essentiality of the (p)ppGpp hydrolase domain of the RSH protein. The staphylococcal RSH-hydrolase is an efficient enzyme that prevents the toxic accumulation of (p)ppGpp. Expression of (p)ppGpp synthases in a hydrolase-negative background leads not only to growth arrest but also to cell death. Transcriptional analyses showed that relP and relQ are strongly induced upon vancomycin and ampicillin treatments. Accordingly, mutants lacking relP and relQ showed a significantly reduced survival rate upon treatments with cell wall-active antibiotics. Thus, RelP and RelQ are active (p)ppGpp synthases in S. aureus that are induced under cell envelope stress to mediate tolerance against these conditions.

• Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Kohler S, Occhialini A
• Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses.
• BMC Genomics. 2013; 14: 459-459
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• BACKGROUND: In the intracellular pathogen Brucella spp., the activation of the stringent response, a global regulatory network providing rapid adaptation to growth-affecting stress conditions such as nutrient deficiency, is essential for replication in the host. A single, bi-functional enzyme Rsh catalyzes synthesis and hydrolysis of the alarmone (p)ppGpp, responsible for differential gene expression under stringent conditions. RESULTS: cDNA microarray analysis allowed characterization of the transcriptional profiles of the B. suis 1330 wild-type and Deltarsh mutant in a minimal medium, partially mimicking the nutrient-poor intramacrophagic environment. A total of 379 genes (11.6% of the genome) were differentially expressed in a rsh-dependent manner, of which 198 were up-, and 181 were down-regulated. The pleiotropic character of the response was confirmed, as the genes encoded an important number of transcriptional regulators, cell envelope proteins, stress factors, transport systems, and energy metabolism proteins. Virulence genes such as narG and sodC, respectively encoding respiratory nitrate reductase and superoxide dismutase, were under the positive control of (p)ppGpp, as well as expression of the cbb3-type cytochrome c oxidase, essential for chronic murine infection. Methionine was the only amino acid whose biosynthesis was absolutely dependent on stringent response in B. suis. CONCLUSIONS: The study illustrated the complexity of the processes involved in adaptation to nutrient starvation, and contributed to a better understanding of the correlation between stringent response and Brucella virulence. Most interestingly, it clearly indicated (p)ppGpp-dependent cross-talk between at least three stress responses playing a central role in Brucella adaptation to the host: nutrient, oxidative, and low-oxygen stress.

• Maisonneuve E, Castro-Camargo M, Gerdes K
• (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity.
• Cell. 2013; 154: 1140-50
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• Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.

• Jin DJ, Cagliero C, Zhou YN
• Role of RNA polymerase and transcription in the organization of the bacterial nucleoid.
• Chem Rev. 2013; 113: 8662-82

• Boutte CC, Crosson S
• Bacterial lifestyle shapes stringent response activation.
• Trends Microbiol. 2013; 21: 174-80
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• Bacteria inhabit enormously diverse niches and have a correspondingly large array of regulatory mechanisms to adapt to often inhospitable and variable environments. The stringent response (SR) allows bacteria to quickly reprogram transcription in response to changes in nutrient availability. Although the proteins controlling this response are conserved in almost all bacterial species, recent work has illuminated considerable diversity in the starvation cues and regulatory mechanisms that activate stringent signaling proteins in bacteria from different environments. In this review, we describe the signals and genetic circuitries that control the stringent signaling systems of a copiotroph, a bacteriovore, an oligotroph, and a mammalian pathogen -Escherichia coli, Myxococcus xanthus, Caulobacter crescentus, and Mycobacterium tuberculosis, respectively - and discuss how control of the SR in these species is adapted to their particular lifestyles.

• Yao Z, Davis RM, Kishony R, Kahne D, Ruiz N
• Regulation of cell size in response to nutrient availability by fatty acid biosynthesis in Escherichia coli.
• Proc Natl Acad Sci U S A. 2012; 109: 25618-25618
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• Cell size varies greatly among different types of cells, but the range in size that a specific cell type can reach is limited. A long-standing question in biology is how cells control their size. Escherichia coli adjusts size and growth rate according to the availability of nutrients so that it grows larger and faster in nutrient-rich media than in nutrient-poor media. Here, we describe how, using classical genetics, we have isolated a remarkably small E. coli mutant that has undergone a 70% reduction in cell volume with respect to wild type. This mutant lacks FabH, an enzyme involved in fatty acid biosynthesis that previously was thought to be essential for the viability of E. coli. We demonstrate that although FabH is not essential in wild-type E. coli, it is essential in cells that are defective in the production of the small-molecule and global regulator ppGpp. Furthermore, we have found that the loss of FabH causes a reduction in the rate of envelope growth and renders cells unable to regulate cell size properly in response to nutrient excess. Therefore we propose a model in which fatty acid biosynthesis plays a central role in regulating the size of E. coli cells in response to nutrient availability.

• Angelini S, My L, Bouveret E
• Disrupting the Acyl Carrier Protein/SpoT interaction in vivo: identification of ACP residues involved in the interaction and consequence on growth.
• PLoS One. 2012; 7: 36111-36111
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• In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation.

• Pal RR, Bag S, Dasgupta S, Das B, Bhadra RK
• Functional characterization of the stringent response regulatory gene dksA of Vibrio cholerae and its role in modulation of virulence phenotypes.
• J Bacteriol. 2012; 194: 5638-48
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• In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA(Vc)), coding for the protein DksA(Vc). Extensive genetic analyses of the DeltadksA(Vc) mutants suggest that DksA(Vc) is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA(Vc) positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA(Vc) upregulates expression of the sigma factor FliA (sigma(28)), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA(Vc) also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

• Tagami K et al.
• Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome.
• Microbiologyopen. 2012; 1: 115-34
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• To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the DeltarelA DeltayjbM DeltaywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

• Manuel J, Berry C, Selin C, Fernando WG, de Kievit TR
• Repression of the antifungal activity of Pseudomonas sp. strain DF41 by the stringent response.
• Appl Environ Microbiol. 2011; 77: 5635-42
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• The stringent response (SR) enables bacteria to adapt to nutrient limitation through production of the nucleotides guanosine tetraphosphate and guanosine pentaphosphate, collectively known as (p)ppGpp. Two enzymes are responsible for the intracellular pools of (p)ppGpp: RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. We investigated how the SR affects the ability of the biological control agent Pseudomonas sp. strain DF41 to inhibit the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary. Strain DF41 relA and relA spoT mutants were generated and found to exhibit increased antifungal activity. Strain DF41 produces a lipopeptide (LP) molecule that is essential for Sclerotinia biocontrol. LP production and protease activity were both elevated in the relA and relA spoT mutants. Addition of relA but not spoT in trans restored the mutant phenotype to that of the parent. Next, we investigated whether an association exists between the SR and known regulators of biocontrol, including the Gac system and RpoS. A gacS mutant of strain DF41 produced less (p)ppGpp and exhibited a 1.7-fold decrease in relA expression compared to the wild type, suggesting that relA forms part of the Gac regulon. We discovered that rpoS transcription was reduced significantly in the SR mutants. Furthermore, rpoS provided in trans restored protease activity to wild-type levels but did not attenuate antifungal activity. Finally, relA expression was decreased in the mutants, indicating that the SR is required for maximum expression of relA.

• Atkinson GC, Tenson T, Hauryliuk V
• The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life.
• PLoS One. 2011; 6: 23479-23479
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• RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.

• Potrykus K, Murphy H, Philippe N, Cashel M
• ppGpp is the major source of growth rate control in E. coli.
• Environ Microbiol. 2011; 13: 563-75
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• It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent, RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp-deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared with fast growing wild-type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp-deficient cells restores this control. We believe these findings strongly argue in favour of ppGpp and against redundant regulation of growth rate control by other factors in Escherichia coli and other enteric bacteria.

• Cavanagh AT, Chandrangsu P, Wassarman KM
• 6S RNA regulation of relA alters ppGpp levels in early stationary phase.
• Microbiology. 2010; 156: 3791-800
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• 6S RNA is a small, non-coding RNA that interacts directly with sigma(70)-RNA polymerase and regulates transcription at many sigma(70)-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from sigma(70)-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of sigma(S) activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions.

• da Silva CA, Balhesteros H, Mazzon RR, Marques MV
• SpdR, a response regulator required for stationary-phase induction of Caulobacter crescentus cspD.
• J Bacteriol. 2010; 192: 5991-6000
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• The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

• Mutalik VK, Nonaka G, Ades SE, Rhodius VA, Gross CA
• Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica.
• J Bacteriol. 2009; 191: 7279-87
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• The sigma(E)-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. sigma(E) is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the sigma(E) regulon is driven from a suite of approximately 60 sigma(E)-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the sigma(E) promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (-35 to -65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the alpha-subunit of RNA polymerase; (ii) ppGpp generally activates sigma(E) promoters, and (iii) sigma(E) promoters are responsive to changing sigma(E) holoenzyme levels under physiological conditions, reinforcing the idea that the sigma(E) regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

• Wout PK, Sattlegger E, Sullivan SM, Maddock JR
• Saccharomyces cerevisiae Rbg1 protein and its binding partner Gir2 interact on Polyribosomes with Gcn1.
• Eukaryot Cell. 2009; 8: 1061-71
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• Rbg1 is a previously uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg/CgtA subfamily of GTP-binding proteins whose members are involved in ribosome function in both prokaryotes and eukaryotes. We show here that Rbg1 specifically associates with translating ribosomes. In addition, in this study proteins were identified that interact with Rbg1 by yeast two-hybrid screening and include Tma46, Ygr250c, Yap1, and Gir2. Gir2 contains a GI (Gcn2 and Impact) domain similar to that of Gcn2, an essential factor of the general amino acid control pathway required for overcoming amino acid shortage. Interestingly, we found that Gir2, like Gcn2, interacts with Gcn1 through its GI domain, and overexpression of Gir2, under conditions mimicking amino acid starvation, resulted in inhibition of growth that could be reversed by Gcn2 co-overexpression. Moreover, we found that Gir2 also cofractionated with polyribosomes, and this fractionation pattern was partially dependent on the presence of Gcn1. Based on these findings, we conclude that Rbg1 and its interacting partner Gir2 associate with ribosomes, and their possible biological roles are discussed.

• Natori Y et al.
• Transcription activity of individual rrn operons in Bacillus subtilis mutants deficient in (p)ppGpp synthetase genes, relA, yjbM, and ywaC.
• J Bacteriol. 2009; 191: 4555-61
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• 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.

• Persky NS, Ferullo DJ, Cooper DL, Moore HR, Lovett ST
• The ObgE/CgtA GTPase influences the stringent response to amino acid starvation in Escherichia coli.
• Mol Microbiol. 2009; 73: 253-66
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• The stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro, implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo. Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.

• Shah S, Das B, Bhadra RK
• Functional analysis of the essential GTP-binding-protein-coding gene cgtA of Vibrio cholerae.
• J Bacteriol. 2008; 190: 4764-71
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• The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by using genetic analysis that cgtA is essential in Vibrio cholerae even in a Delta relA background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to the replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of CgtA plays a critical role in its proper function.

• Nascimento MM, Lemos JA, Abranches J, Lin VK, Burne RA
• Role of RelA of Streptococcus mutans in global control of gene expression.
• J Bacteriol. 2008; 190: 28-36
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• The production of (p)ppGpp by Streptococcus mutans UA159 is catalyzed by three gene products: RelA, RelP, and RelQ. Here, we investigate the role of the RelA (Rel) homologue of S. mutans in the stringent response and in the global control of gene expression. RelA of S. mutans was shown to synthesize pppGpp in vitro from GTP and ATP in the absence of added ribosomes, as well as in vivo in an Escherichia coli relA-spoT mutant. Mupirocin (MUP) was shown to induce high levels of (p)ppGpp production in S. mutans in a relA-dependent manner, with a concomitant reduction in GTP pools. Transcription profiling after MUP treatment of S. mutans revealed that 104 genes were upregulated and 130 were downregulated (P < or = 0.001); mainly, genes for macromolecular biosynthesis, translation, and energy metabolism were downregulated. When a derivative of UA159 carrying a complete deletion of the relA gene was treated with MUP, 72 genes were upregulated and 52 were downregulated (P < or = 0.001). The expression of 50 genes (P < or = 0.001) was commonly affected by MUP treatment in the two strains, suggesting that S. mutans can mount a relA-independent response to MUP. Consistent with the gene expression profiling, RelA was shown to play major roles in the regulation of phenotypic traits that are required for establishment, persistence, and virulence expression by this oral pathogen. Thus, RelA is the major (p)ppGpp synthase controlling the stringent response in S. mutans, and it coordinates the expression of genes and phenotypes that contribute to the pathogenic potential of the organism.

• Masuda S, Tozawa Y, Ohta H
• Possible targets of "magic spots" in plant signalling.
• Plant Signal Behav. 2008; 3: 1021-3
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• The prokaryotic signalling molecules (p)ppGpp, also called "magic spots", regulate a wide variety of physiological activities in bacteria, including transcription, translation, and replication as well as some enzymatic activities such as those of some GTP-binding proteins, which are necessary for bacterial cells to adapt their physiology to different environmental stimuli. This response is called the stringent response. Recently, (p)ppGpp molecules and (p)ppGpp synthetase homologues, designated RSHs, have been identified in plants. At least some of the RSHs are targeted to chloroplasts. A knockdown mutation in one of the RSHs results in unusual flower development in Arabidopsis, suggesting that the plastid stringent response has important roles in the physiology of higher plants. Possible (p)ppGpp target proteins are investigated.

• Lemos JA, Nascimento MM, Lin VK, Abranches J, Burne RA
• Global regulation by (p)ppGpp and CodY in Streptococcus mutans.
• J Bacteriol. 2008; 190: 5291-9
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• 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 (DeltarelAPQ) 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 DeltarelAPQ strain fully restored growth in medium lacking leucine or valine, revealing that the growth-defective phenotype of DeltarelAPQ 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.

• Traxler MF et al.
• The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.
• Mol Microbiol. 2008; 68: 1128-48
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• The stringent response to amino acid starvation, whereby stable RNA synthesis is curtailed in favour of transcription of amino acid biosynthetic genes, is controlled by the alarmone ppGpp. To elucidate the extent of gene expression effected by ppGpp, we designed an experimental system based on starvation for isoleucine, which could be applied to both wild-type Escherichia coli and the multiauxotrophic relA spoT mutant (ppGpp(0)). We used microarrays to profile the response to amino acid starvation in both strains. The wild-type response included induction of the general stress response, downregulation of genes involved in production of macromolecular structures and comprehensive restructuring of metabolic gene expression, but not induction of amino acid biosynthesis genes en masse. This restructuring of metabolism was confirmed using kinetic Biolog assays. These responses were profoundly altered in the ppGpp(0) strain. Furthermore, upon isoleucine starvation, the ppGpp(0) strain exhibited a larger cell size and continued growth, ultimately producing 50% more biomass than the wild-type, despite producing a similar amount of protein. This mutant phenotype correlated with aberrant gene expression in diverse processes, including DNA replication, cell division, and fatty acid and membrane biosynthesis. We present a model that expands and functionally integrates the ppGpp-mediated stringent response to include control of virtually all macromolecular synthesis and intermediary metabolism.

• Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L
• What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?
• J Bacteriol. 2007; 189: 6101-8
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• The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.

• Ossa F et al.
• The Myxococcus xanthus Nla4 protein is important for expression of stringent response-associated genes, ppGpp accumulation, and fruiting body development.
• J Bacteriol. 2007; 189: 8474-83
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• Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.

• Magnusson LU, Gummesson B, Joksimovic P, Farewell A, Nystrom T
• Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli.
• J Bacteriol. 2007; 189: 5193-202
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• The recent discovery that the protein DksA acts as a coregulator of genes controlled by ppGpp led us to investigate the similarities and differences between the relaxed phenotype of a ppGpp-deficient mutant and the phenotype of a strain lacking DksA. We demonstrate that the absence of DksA and ppGpp has similar effects on many of the observed phenotypes but that DksA and ppGpp also have independent and sometimes opposing roles in the cell. Specifically, we show that overexpression of DksA can compensate for the loss of ppGpp with respect to transcription of the promoters P(uspA), P(livJ), and P(rrnBP1) as well as amino acid auxotrophy, cell-cell aggregation, motility, filamentation, and stationary phase morphology, suggesting that DksA can function without ppGpp in regulating gene expression. In addition, ppGpp and DksA have opposing effects on adhesion. In the course of our analysis, we also discovered new features of the relaxed mutant, namely, defects in cell-cell aggregation and motility.

• Silva AJ, Benitez JA
• A Vibrio cholerae relaxed (relA) mutant expresses major virulence factors, exhibits biofilm formation and motility, and colonizes the suckling mouse intestine.
• J Bacteriol. 2006; 188: 794-800
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• We have constructed a relaxed mutant of El Tor biotype Vibrio cholerae strain C7258 by disruption of the RelA catalytic domain. The ability of the V. cholerae relaxed mutant to biosynthesize guanosine tetraphosphate and pentaphosphate was severely affected; the mutant showed a reduced growth rate in minimal medium that could be reversed by the addition of Casamino Acids, and it was thermosensitive. Contrary to published findings, the new relA mutant still produced significant cholera toxin and toxin-coregulated pilus. The V. cholerae relA mutant was motile, produced normal biofilms, and colonized the suckling mouse intestine. Our data suggest that levels of basal guanosine nucleotides pppGpp and ppGpp, rather than the availability of a stringent response, could influence expression of virulence factors, depending on strain and culture conditions. Production of hemagglutinin (HA)/protease, which requires HapR, RpoS, and the cyclic AMP receptor protein, was not strongly affected. Nevertheless, overexpression of RelA protein from an isopropyl-beta-d-thiogalactopyranoside-inducible promoter posttranscriptionally diminished production of HA/protease.

• Wells DH, Gaynor EC
• Helicobacter pylori initiates the stringent response upon nutrient and pH downshift.
• J Bacteriol. 2006; 188: 3726-9
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• Helicobacter pylori was previously reported to lack a stringent response. In contrast, we show that after nutrient downshift, H. pylori produced abundant ppGpp and less total RNA. pH downshift also caused (p)ppGpp accumulation. Our observations indicate that nutrient deprivation and acid shock activate the stringent response in H. pylori.

• Jain V, Saleem-Batcha R, China A, Chatterji D
• Molecular dissection of the mycobacterial stringent response protein Rel.
• Protein Sci. 2006; 15: 1449-64
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• Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.

• Mouery K, Rader BA, Gaynor EC, Guillemin K
• The stringent response is required for Helicobacter pylori survival of stationary phase, exposure to acid, and aerobic shock.
• J Bacteriol. 2006; 188: 5494-500
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• The gastric pathogen Helicobacter pylori must adapt to fluctuating conditions in the harsh environment of the human stomach with the use of a minimal number of transcriptional regulators. We investigated whether H. pylori utilizes the stringent response, involving signaling through the alarmone (p)ppGpp, as a survival strategy during environmental stresses. We show that the H. pylori homologue of the bifunctional (p)ppGpp synthetase and hydrolase SpoT is responsible for all cellular (p)ppGpp production in response to starvation conditions. Furthermore, the H. pylori spoT gene complements the growth defect of Escherichia coli mutants lacking (p)ppGpp. An H. pylori spoT deletion mutant is impaired for stationary-phase survival and undergoes a premature transformation to a coccoid morphology. In addition, the spoT deletion mutant is unable to survive specific environmental stresses, including aerobic shock and acid exposure, which are likely to be encountered by this bacterium during infection and transmission.

• Kasai K, Nishizawa T, Takahashi K, Hosaka T, Aoki H, Ochi K
• Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus.
• J Bacteriol. 2006; 188: 7111-22
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• Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

• Dahl JL et al.
• The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression.
• J Bacteriol. 2005; 187: 2439-47
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• The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.

• Kasai K, Kanno T, Endo Y, Wakasa K, Tozawa Y
• Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline.
• Nucleic Acids Res. 2004; 32: 5732-41
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• Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria.

• Erickson DL, Lines JL, Pesci EC, Venturi V, Storey DG
• Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.
• Infect Immun. 2004; 72: 5638-45
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• The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

• Silva AJ, Benitez JA
• Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS.
• J Bacteriol. 2004; 186: 6374-82
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• Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities. Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR. In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases. Addition of glucose in these phases repressed hapA transcription by inducing V. cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion. Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded sigma(s) factor. The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase. Analysis of rpoS and hapR mRNA in isogenic CRP+ and CRP- strains suggested that CRP enhances the transcription of rpoS and hapR. Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli. Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression. Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter. This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors.

• Bugrysheva J et al.
• Characterization of the stringent response and rel(Bbu) expression in Borrelia burgdorferi.
• J Bacteriol. 2003; 185: 957-65
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• The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis. rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA. These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.

• Yamada A, Tsutsumi K, Tanimoto S, Ozeki Y
• Plant RelA/SpoT homolog confers salt tolerance in Escherichia coli and Saccharomyces cerevisiae.
• Plant Cell Physiol. 2003; 44: 3-9
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• To analyze the mechanisms of salt tolerance in the halophyte Suaeda japonica, Escherichia coli was used as a host organism to undertake functional screening of cDNAs encoding proteins that may play an important role for the salt-tolerance mechanisms. A transformant expressing RelA/SpoT homolog (Sj-RSH) was found to have enhanced salt tolerance. In E. coli, RelA/SpoT controlled the amount of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effectors of the bacterial stringent response. Complementation analysis using the relA mutant of E. coli showed that Sj-RSH conferred the phenotype associated with (p)ppGpp synthesis. Furthermore, expression of Sj-RSH driven by the GAL1 promoter also gave rise to enhanced salt tolerance in yeast. Northern blot analyses of the yeast transformant revealed that the transcriptional levels of stress responsive genes including GPD1, VMA6, BMH1, HYP1 and HOG1 were clearly enhanced in the Sj-RSH transformant when compared with an empty vector transformant under stress-free and 1.5 M NaCl stress conditions. These results suggest that (p)ppGpp synthesis mediated by plant RelA/SpoT homologs plays a critical role for the transcriptional induction of several stress responsive genes, directly or indirectly in yeast, and that the conserved stress-resistance system may exist in higher plants.

• Jishage M, Kvint K, Shingler V, Nystrom T
• Regulation of sigma factor competition by the alarmone ppGpp.
• Genes Dev. 2002; 16: 1260-70
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• Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

• Taylor CM et al.
• Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence.
• J Bacteriol. 2002; 184: 621-8
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• We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.

• Brown L, Gentry D, Elliott T, Cashel M
• DksA affects ppGpp induction of RpoS at a translational level.
• J Bacteriol. 2002; 184: 4455-65
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• The RpoS sigma factor (also called sigmaS or sigma38) is known to regulate at least 50 genes in response to environmental sources of stress or during entry into stationary phase. Regulation of RpoS abundance and activity is complex, with many factors participating at multiple levels. One factor is the nutritional stress signal ppGpp. The absence of ppGpp blocks or delays the induction of rpoS during entry into stationary phase. Artificially inducing ppGpp, without starvation, is known to induce rpoS during the log phase 25- to 50-fold. Induction of ppGpp is found to have only minor effects on rpoS transcript abundance or on RpoS protein stability; instead, the efficiency of rpoS mRNA translation is increased by ppGpp as judged by both RpoS pulse-labeling and promoter-independent effects on lacZ fusions. DksA is found to affect RpoS abundance in a manner related to ppGpp. Deleting dksA blocks rpoS induction by ppGpp. Overproduction of DksA induces rpoS but not ppGpp. Deleting dksA neither alters regulation of ppGpp in response to amino acid starvation nor nullifies the inhibitory effects of ppGpp on stable RNA synthesis. Although this suggests that dksA is epistatic to ppGpp, inducing ppGpp does not induce DksA. A dksA deletion does display a subset of the same multiple-amino-acid requirements found for ppGpp(0) mutants, but overproducing DksA does not satisfy ppGpp(0) requirements. Sequenced spontaneous extragenic suppressors of dksA polyauxotrophy are frequently the same T563P rpoB allele that suppresses a ppGpp(0) phenotype. We propose that DksA functions downstream of ppGpp but indirectly regulates rpoS induction.

• Barker MM, Gaal T, Josaitis CA, Gourse RL
• Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro.
• J Mol Biol. 2001; 305: 673-88
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• To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.

• Gropp M, Strausz Y, Gross M, Glaser G
• Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain.
• J Bacteriol. 2001; 183: 570-9
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• The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA(+) cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752-5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

• Carmona M, Rodriguez MJ, Martinez-Costa O, De Lorenzo V
• In vivo and in vitro effects of (p)ppGpp on the sigma(54) promoter Pu of the TOL plasmid of Pseudomonas putida.
• J Bacteriol. 2000; 182: 4711-8
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• The connection between the physiological control of the sigma(54)-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA. Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the relA gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in Pu activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in Pu performance through a range of sigma(54)-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of sigma(54)-systems.

• Gentry D, Li T, Rosenberg M, McDevitt D
• The rel gene is essential for in vitro growth of Staphylococcus aureus.
• J Bacteriol. 2000; 182: 4995-7
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• The stringent response in Staphylococcus aureus is mediated by the nucleotide guanosine pentaphosphate, whose synthesis is catalyzed by the product of the rel gene. We report here that the rel gene is essential for the in vitro growth of S. aureus, distinguishing it from all other bacteria tested for this requirement.

• Martinez-Costa OH, Fernandez-Moreno MA, Malpartida F
• The relA/spoT-homologous gene in Streptomyces coelicolor encodes both ribosome-dependent (p)ppGpp-synthesizing and -degrading activities.
• J Bacteriol. 1998; 180: 4123-32
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• Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

• Zhou YN, Jin DJ
• RNA polymerase beta mutations have reduced sigma70 synthesis leading to a hyper-temperature-sensitive phenotype of a sigma70 mutant.
• J Bacteriol. 1997; 179: 4292-8
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• This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

• Khattar MM, Addinall SG, Stedul KH, Boyle DS, Lutkenhaus J, Donachie WD
• Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function.
• J Bacteriol. 1997; 179: 784-93
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• Two new mutations in the cell division gene ftsW have been isolated and characterized. The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation. The ftsW1640(Ts) mutation, however, causes a block to division at a later stage. The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent. Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site. Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure. We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations.

• Zhang X, Bremer H
• Control of the Escherichia coli rrnB P1 promoter strength by ppGpp.
• J Biol Chem. 1995; 270: 11181-9
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• Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2 combination, to a wild-type lacZ gene were constructed on plasmids and recombined into the mal region of the bacterial chromosome, close to the normal location and in the normal orientation of rrnB. The upstream activator region (Fis-binding sites) was always present with the P1 promoter, and all constructs contained the box A antitermination site of rRNA genes. Using these constructs, beta-galactosidase specific activities were measured in Escherichia coli strains carrying either both ppGpp synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+), or neither (delta relA delta spoT), using different media supporting growth rates between 0.6 and 2.8 doublings/h at 37 degrees C. The beta-galactosidase activities were used to estimate the relative strength of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter. Promoter strength (transcripts initiated per min per promoter per free RNA polymerase concentration) was distinguished from promoter activity (transcripts initiated per min per promoter). In ppGpp-synthesizing (wild-type) bacteria, the relative strength of the rrnB P1 promoter increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus, at low or zero levels of ppGpp, the P1 promoter was 1.5-1.7 times stronger than the isolated P2 promoter. These results indicate that the normal growth rate control of the rrnB P1 promoter strength requires ppGpp, and that the strength is reduced at basal levels of ppGpp found during exponential growth. No additional ppGpp-independent control of the rrnB P1 promoter strength was evident. From the beta-galactosidase data and previously determined values of rRNA gene activities, the activities of the isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem combination, were estimated. With increasing growth rate, the activity of the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min, while the activity of the isolated P1 promoter increased 24-fold from 2 to 54 initiations/min. The increasing activity of the isolated P2 promoter is assumed to reflect the increasing RNA polymerase concentration at constant promoter strength, whereas the steeper increase in P1 promoter activity reflects increases in both polymerase concentration and promoter strength. When in tandem with P1, the P2 promoter activity is inferred to decrease as the P1 promoter activity increases.

• Faxen M, Isaksson LA
• Functional interactions between translation, transcription and ppGpp in growing Escherichia coli.
• Biochim Biophys Acta. 1994; 1219: 425-34
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• Strains with a relA mutation together with three different alleles of spoT were used to study the effects of different levels of ppGpp on production time for beta-galactosidase, transcriptional polarity and readthrough of a stop codon by near-cognate tRNA or a suppressor tRNA. The influences of an rpsL(S12) allele and a miaA mutation, together giving decreased efficiency of translation, as well as an rpoB mutation, coding for an altered RNA polymerase, were also investigated. The spoT alleles which give total deficiency for ppGpp, or a level which is increased several-fold (Sarubbi et al. (1988) Mol. Gen. Genet. 213, 214-222), had at the most a marginal effect on the production time for a beta-galactosidase molecule or translational misreading of a nonsense mutation. The efficiency of an amber tRNA suppressor is not affected by ppGpp in strains with an otherwise wildtype translational machinery. These data suggest that ppGpp does not influence directly the translational process in vivo. Instead, ppGpp is found to interfere with transcriptional readthrough in a manner which is dependent on the rpsL224, miaA, as well as the rpoB mutations. Similarly, bacterial growth is affected by ppGpp in a manner which is dependent on properties of both the transcriptional and translational apparatus together. It is suggested that the primary effect of ppGpp is on transcriptional readthrough, but this effect is modified by translational/transcriptional coupling.

• Lloyd RG, Sharples GJ
• Molecular organization and nucleotide sequence of the recG locus of Escherichia coli K-12.
• J Bacteriol. 1991; 173: 6837-43
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• The nucleotide sequence of the Escherichia coli K-12 recG gene was determined. recG was identified as an open reading frame located between the spoT operon and the convergent gltS gene. It encodes a polypeptide of 693 amino acids which was identified as a 76-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after it was labeled with [35S]methionine in maxicells. The sequence determined revealed no obvious promoter. Synthesis of RecG by plasmids carrying the intact gene varied with the orientation of the insert relative to the vector promoter and with the extent of upstream spoT operon sequence included in the construction. It is concluded that recG is the fourth and last gene in the spoT operon, although a possible promoter for independent transcription of spoU and recG was identified near the end of the spoT gene. The primary sequence of RecG revealed that it is related to proteins that act as helicases and has a well-conserved motif identified with ATP binding.

• Hecker M, Schroeter A, Mach F
• Physiological studies on pBR 322 DNA amplification in an Escherichia coli relA strain.
• J Basic Microbiol. 1986; 26: 329-33
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• Amino acid limitation leads in E. coli relA cells which cannot synthesize guanosine tetraphosphate (ppGpp) under these conditions to an amplification of pBR 322 DNA. We previously proposed that ppGpp produced in E. coli relA+ cells subjected to amino acid limitation inhibits pBR 322 DNA replication (Hecker et al. 1983). In further experiments it was established that an E. coli relA strain shows plasmid amplification during amino acid limitation (arginine, threonine, leucine or histidine) only in the presence of sufficient concentrations of phosphate, ammonia and glucose. Plasmid amplification does not occur if ammonia or phosphate is depleted. We suggest that glucose, ammonia and phosphate are needed for nucleotide and deoxynucleotide synthesis as the essential prerequisite for plasmid amplification. The activity of beta-lactamase was determined as an indicator for the expression of plasmid-encoded genes. The enzyme activity remains on a low level during plasmid amplification because of arginine exhaustion. A remarkable increase in the activity of beta-lactamase was observed after resumption of growth of relA cells containing amplified plasmid DNA. The plasmid content decreased as the cells continued to grow. We found that plasmid amplification and expression of plasmid-localized genes are opposite reactions which do not occur at the same time.

• Gordeev VK, Turkov MI
• [Expression of the amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus. IV. The effect of mutations disturbing the coupling of the transcription and translation processes on ilv-operon expression in cells carrying the mutation in the spoT gene].
• Genetika. 1983; 19: 1433-8
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• The rate of adaptation of Escherichia coli K-12 NF930 spoT1 cells with elevated intracellular level of ppGpp to various minimal media was studied. It has been found that the rate of adaptation of spoT cells, like that of parent and rel strains, depends mainly on the rate of derepression of the ilv operon. The maximal rate of the ilv operon derepression was observed when an optimal concentration of ppGpp was maintained in cells. Derepression of the ilv operon is sharply delayed when the level of ppGpp is elevated or reduced. Mutations altering the translation system do not change the rate of adaptation of spoT cells. Rifampicin resistance mutations which altered the structure of RNA polymerase change the rate of adaptation of spoT cells to minimal media, especially to those containing serine at high concentrations. The possible role of serine in the regulation of ppGpp degradation system is discussed.

• Kliachko EV, Bochkanov SS, Shakulov RS
• [Metabolic regulation of threonine operon transcription in E. coli cells].
• Biokhimiia. 1983; 48: 1095-102
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• The threonine biosynthetic operon transcription in E. coli cells during balanced growth was studied. The rate of threonine-mRNA synthesis was measured by hybridization of impulse-labelled RNA with pYN 1107 DNA carrying the structural threonine genes A, B, C. It was shown that threonine-mRNA synthesis depends on bacterial growth rate being maximal at mu = 0.8 doublings per hour. The influence of the ppGpp on the bacterial growth rate and threonine-mRNA synthesis rate was demonstrated, using spoT-mutants and strains with several relA gene copies. The rate of threonine-mRNA synthesis is maximal at the ppGpp level of about 50-60 pmole/A450. The deviation from this ppGpp optimum level results in inhibition of the threonine-mRNA synthesis. Thus, ppGpp appears to be involved in metabolic regulation of operon transcription. A mechanism of negative regulation of threonine-mRNA synthesis by high concentrations of ppGpp is discussed.

• Turnbough CL Jr
• Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates.
• J Bacteriol. 1983; 153: 998-1007
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• The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed.

• Friesen JD, An G, Fiil NP
• Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.
• Cell. 1978; 15: 1187-97
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• We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.

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