Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

Synthesis and hydrolysis of pppGpp in mycobacteria: a ligand mediated conformational switch in Rel

Bacteria respond to starvation by synthesizing a polyphosphate derivative of guanosine, (p)ppGpp, that helps the bacteria in surviving during stress. The protein in Gram-positive organisms required for (p)ppGpp synthesis is Rel, a bifunctional enzyme that carries out both synthesis and hydrolysis of this molecule. Rel shows increased pppGpp synthesis in the presence of uncharged tRNA, the effect of which is regulated by the C-terminal of Rel. We show by fluorescence resonance energy transfer that the distance between the N-terminus cysteine residue at the catalytic domain and C692 at the C-terminus increases upon the addition of uncharged tRNA. In apparent anomaly, the steady state anisotropy of the Rel protein decreases upon tRNA binding suggesting "compact conformation" vis-à-vis "open conformation" of the free Rel. We propose that the interaction between C692 and the residues present in the pppGpp synthesis site results in the regulated activity and this interaction is abrogated upon addition of uncharged tRNA. We also report here the binding of pppGpp to the C-terminal part of the protein that leads to more unfolding in this region.     

Replacement of pseudouridine in transfer RNA by 5-fluorouridine does not affect the ability to stimulate the synthesis of guanosine 5'-triphosphate 3'-diphosphate.

The requirement for ribothymidine and pseudouridine in the TpsiCG loop of tRNA for its activity in the ribosome and tRNA-stimulated synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) by stringent factor has been tested by the use of a purified tRNAPhe (883 pmol of phenylalanine incorporated/A260 unit) in which 92% of the pseudouridine, 98% of the ribothymidine, 98% of the dihydrouridine, and 88% of the uridines were substituted by 5-fluorouridine. This tRNA was quantitatively as active as control tRNA in inducing pppGpp synthesis. With loose-couple ribosomes, the concentration of tRNA needed to give half-maximal reaction was 0.07 micrometer for both normal and fluorouridine-substituted tRNA, with vacant tight-couple ribosomes it was 0.05 micrometer, and with tight couples carrying poly(Phe)-tRNA at the P site the value was 0.02 micrometer. These results show that at the level of intact tRNA there is no special requirement for modified bases in the TpsiCG loop of tRNA in the synthesis of pppGpp.     

Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

We have investigated the changes in the guanosine 5'-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5'-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5'-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5'-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.     

Peptide‐nucleotide antibiotic Microcin C is a potent inducer of stringent response and persistence in both sensitive and producing cells

Microcin C (McC) is a peptide‐nucleotide antibiotic that inhibits aspartyl‐tRNA synthetase. Here, we show that McC is a strong inducer of persistence in Escherichia coli. Persistence induced by McC is mediated by (p)ppGpp and requires chromosomally encoded toxin‐antitoxin modules. McC‐producing cells have increased persistence levels due to a combined effect of McC imported from the cultured medium and intracellularly synthesized antibiotic. McC‐producing cells also induce persistence in sensitive cells during co‐cultivation, underscoring complex interactions in bacterial communities where an antagonistic compound produced by one community member can benefit other members by increasing their ability to withstand antibiotics.     

Guanosine polyphosphate concentration and stable RNA synthesis in Bacillus subtilis following suppression of protein synthesis

Amount of guanosine-5'-triphosphate, 3'-diphosphate (pppGpp) and guanosine-5'-diphosphate, 3'-diphosphate (ppGpp) in the cells of b. subtilis increased several times during starvation for lysine or after treatment with serine hydroxamate (analog of serine) or norvaline (analog of leucine), or in the presence of trimethoprim, which induced deficiency of methionine and leucine. In exponentially growing cells the concentration of pppGpp was found to be 10-20 pmol/A600. When serine hydroxamate or trimethoprim were added, concentration of pppGpp increased to 500-800 pmol/A600 and then slowly diminished. Elimination of lysine or addition to the culture medium of norvaline caused slight transitory accumulation of pppGpp (150 pmol/A600). The amount of another nucleotide ppGpp was always 2-3 times lower than one of pppGpp. Accumulation of (p)ppGpp in rel+ cells was accompanied by cessation of stable RNA synthesis. Under conditions described above rel- cells continued RNA synthesis and did not accumulate (p)ppGpp. In the rel+ cells treated with serine hydroxamate synthesis of stable RNA resumed and the amount of (p)ppGpp decreased after addition of serine or tetracycline and chloramphenicol. The half-life period for pppGpp in the presence of chloramphenicol was determined to be 30-40 seconds. Thus, during aminoacyl-tRNA deficiency rel+ cells of B. subtilis accumulate (p)ppGpp, which are believed to participate in negative regulation of RNA synthesis. Slight accumulation of pppGpp without concomitant inhibition of stable RNA synthesis was observed after treatment of growing cells with chloramphenicol.     

Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci.

The stringent response in Escherichia coli and many other organisms is regulated by the nucleotides ppGpp and pppGpp. We show here for the first time that at least six staphylococcal species also synthesize ppGpp and pppGpp upon induction of the stringent response by mupirocin. Spots corresponding to ppGpp and pppGpp on thin-layer chromatograms suggest that pppGpp is the principal regulatory nucleotide synthesized by staphylococci in response to mupirocin, rather than ppGpp as in E. coli.     

Purpuromycin: an antibiotic inhibiting tRNA aminoacylation.

Purpuromycin, an antibiotic produced by Actinoplanes ianthinogenes, had been reported previously to inhibit protein synthesis. In the present report, we demonstrate that the mechanism of action of this antibiotic is quite novel in that it binds with fairly high affinity to all tRNAs, inhibiting their acceptor capacity. Although more than one molecule of purpuromycin is bound to each tRNA molecule, the inhibitory activity of this antibiotic was found to be selective for the tRNA acceptor function; in fact, after the aminoacylation step, purpuromycin was found to affect none of the other tested functions of tRNA (interaction with the ribosomal P- and A-sites and interaction with translation factors). Accordingly, purpuromycin was found to inhibit protein synthesis only when translation depended on the aminoacylation of tRNA and not when the system was supplemented with pre-formed aminoacyl-tRNAs. Because purpuromycin did not interfere with the ATP-PPi exchange reaction of the synthetase or with the initial interaction of the enzyme with its tRNA substrate, the basis for the inhibition of aminoacylation is presumably the formation of a nonproductive synthetase-tRNA complex in the presence of purpuromycin in which the tRNA is unable to be charged with the corresponding amino acid.     

Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.     

Stringency and relaxation among the halobacteria.

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.     

Purification and properties of stringent factor.

The stringent factor from Escherichia coli is the product of the relA locus. It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP). This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation. In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e. the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates. Here we report the purification of the stringent factor to near homogeneity. It is a monomeric protein with a molecular weight of 75,000. The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor.     

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.     

Mutants of Escherichia coli defective in the degradation of guanosine 5''-triphosphate, 3''-diphosphate (pppGpp).

Summary A new class of mutants of E. coli exhibiting altered metabolism of ppGpp and pppGpp has been isolated, and mapped at a locus designated gpp, near min 83 on the genetic map. These mutants accumulate elevated levels of pppGpp during amino acid starvation or carbon source downshift, and exhibit a reduced rate of pppGpp degradation in vivo. The in vitro evidence suggests that the gpp mutants are defective in a 5-nucleotidase, which specifically hydrolyzes pppGpp to ppGpp. Certain combinations of gpp and spoT mutations are inviable. A gpp spoT double mutant, constructed by employing a leaky spoT mutation, was found to have a slower rate of pppGpp degradation than the gpp mutant alone. This result indicates that spoT also participates in pppGpp degradation. The inviability of certain gpp spoT combinations is attributed to the inability of the double mutants to degrade pppGpp. This is supported by the observation that selection for increased growth rate on the double mutant results in the recovery of relA mutations. Various effects of the gpp mutation upon the pppGpp and ppGpp pools provide additional support for a scheme in which pppGpp is the major precursor of ppGpp.     

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.     

The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates.

The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T. (1992) J. Biol. Chem. 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase. In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP. Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components. The action of this enzyme is relatively specific for the N-acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N-acylaminoacyl-tRNA, or aminoacyl-tRNA. The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP. The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f[3H]Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c-c). Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA. This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced.     

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.     

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.     

N-thiolated beta-lactams: novel antibacterial agents for methicillin-resistant Staphylococcus aureus

In this report we describe a new family of N-thiolated beta-lactams that have antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The compounds show unprecedented structure-activity features and an unusual mode of action for a beta-lactam antibiotic.