The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus.
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Salmonella enterica serovar Typhimurium (S. typhimurium) is an enteric pathogen that causes significant morbidity in humans and other mammals. During their life cycle, salmonellae must survive frequent exposures to a variety of environmental stresses, e.g. carbon-source (C) starvation. The starvation-stress response (SSR) of S. typhimurium encompasses the genetic and physiological realignments that occur when an essential nutrient becomes limiting for bacterial growth. The function of the SSR is to produce a cell capable of surviving long-term starvation. This paper reports that three C-starvation-inducible lac fusions from an S. typhimurium C-starvation-inducible lac fusion library are all within a gene identified as fadF, which encodes an acyl-CoA dehydrogenase (ACDH) specific for medium-/long-chain fatty acids. This identification is supported by several findings: (a) significant homology at the amino acid sequence level with the ACDH enzymes from other bacteria and eukaryotes, (b) undetectable beta-oxidation levels in fadF insertion mutants, (c) inability of fad insertion mutants to grow on oleate or decanoate as a sole C-source, and (d) inducibility of fadF::lac fusions by the long-chain fatty acid oleate. In addition, the results indicate that the C-starvation-induction of fadF is under negative control by the FadR global regulator and positive control by the cAMP:cAMP receptor protein complex and ppGpp. It is also shown that the fadF locus is important for C-starvation-survival in S. typhimurium. Furthermore, the results demonstrate that fadF is induced within cultured Madin-Darby canine kidney (MDCK) epithelial cells, suggesting that signals for its induction (C-starvation and/or long-chain fatty acids) may be present in the intracellular environment encountered by S. typhimurium. However, fadF insertion mutations did not have an overt effect on mouse virulence. (+info)
Incorporation of 32Pi into nucleotides, polyphosphates, and other acid-soluble compounds by Myxococcus xanthus during myxospore formation.
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When glycerol was used to induce myxospore formation in Myxococcu xanthus in the presence of 32Pi, the label was incorporated into a variety of acid-soluble compounds. Incorporation into ribonucleotides was approximately fivefold greater than in vegetative cells or noninducible mutants grown in glycerol. The label was also incorporated into some unknown compounds and material tentatively identified as guanosine tetraphosphate. Marked accumulation into polyphosphates, which were present mainly in culture supernatants, occurred relatively late during myxospore formation. The kinetics of accumulation of some of these compounds and their distribution into acid-soluble cell extracts and culture supernatants are described and compared with those in vegetative cells and noninducible mutants. (+info)
Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.
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Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP. (+info)
Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.
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In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody. (+info)
Thiostrepton-resistant mutants exhibit relaxed synthesis of RNA.
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Spontaneous mutants of Bacillus subtilis resistant to thiostrepton (TSP) exhibit relaxed synthesis of RNA when starved for required amino acids. Intact cells of tsp mutants cannot synthesize the regulatory nucleotides, ppGpp and pppGpp, after amino acid deprivation. Because ribosomes isolated from spontaneous revertants to thiostrepton sensitivity and from wild-type stringent strains can synthesize (p)ppGpp whereas ribosomes isolated from tsp strains cannot synthesize these regulatory nucleotides in the presence of stringent factor, it appears that the lesion is expressed at the level of the ribosome. Genetic mapping, via three-factor transformational crosses, has shown that tsp is closely linked to rif, in the order cysA14, tsp, rif-I, strA. The phenotype of the tsp mutants indicates that they are of the relC type. Their map position indicates that they are different from a previously described B subtilis rel mutation. Ribosomes from the latter strain can synthesize (p)ppGpp in cell-free extracts. (+info)
Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli.
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Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter. (+info)
Regulation of bacteriophage lambda development by guanosine 5'-diphosphate-3'-diphosphate.
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On infection of its host, Escherichia coli, bacteriophage lambda can follow one of two alternative developmental pathways: lytic or lysogenic. Here we demonstrate that the "lysis-versus-lysogenization" decision is influenced by guanosine tetraphosphate (ppGpp), a nucleotide that is synthesized in E. coli cells in response to amino acid or carbon source starvation. We found that the efficiency of lysogenization is the highest at ppGpp concentrations somewhat higher than the basal level; too low and too high levels of ppGpp result in less efficient lysogenization. Maintenance of the already integrated lambda prophage and phage lytic development were not significantly influenced in the host lacking ppGpp. We found that the level of HflB/FtsH protease, responsible for degradation of the CII protein, an activator of "lysogenic" promoters, depends on ppGpp concentration. The highest levels of HflB/FtsH was found in bacteria lacking ppGpp and in cells bearing increased concentrations of this nucleotide. Using lacZ fusions, we investigated the influence of ppGpp on activities of lambda promoters important at the stage of the lysis-versus-lysogenization decision. We found that each promoter is regulated differentially in response to the abundance of ppGpp. Moreover, our results suggest that the cAMP level may influence ppGpp concentration in cells. The mechanism of the ppGpp-mediated control of lambda development at the stage of the lysis-versus-lysogenization decision may be explained on the basis of differential influence of guanosine tetraphosphate on activities of p(L), p(R), p(E), p(I), and p(aQ) promoters and by dependence of HflB/FtsH protease level on ppGpp concentration. (+info)
Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry.
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The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-). (+info)