A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.     

Enzymes producing 4-thiouridine in Escherichia coli tRNA: approximate chromosomal locations of the genes and enzyme activities in a 4-thiouridine-deficient mutant.

A previously described mutant of Escherichia coli which lacks 4-thiouridine in its tRNA was here shown to be deficient in factor A, one of the two proteins responsible for this thiolation of uridine. Addition of exogenous factor A restored the thiolating ability of extracts prepared from the mutant. The activities of the two thiolation proteins were governed by genes at two widely separated positions on the chromosome, as determined with F-prime merodiploids. The site governing factor A activity lay roughly in the region of the recently reported position of nuv, a gene controlling the production of 4-thiouridine in tRNA.     

Guanosine 5'-diphosphate 3'-diphosphate levels, carbon source, and ribonucleic acid synthesis in a mutant strain of Escherichia coli

We have previously described a mutant strain of Escherichia coli (2S142) which shows a specific inhibition of stable RNA synthesis at 42 degrees C. The temperature-sensitive lesion mimics a carbon source downshift (diauxie lag). We therefore measured RNA synthesis and levels of ppGpp (guanosine 5'-diphosphate 3'-diphosphate) on a number of different carbon sources. There is a 6-fold variation in ppGpp levels at 42 degrees C, depending on the carbon source present. Much of the variation in ppGpp levels at 42 degrees C can be explained by variations in the decay rate of ppGpp at 42 degrees C. The rates of ribosomal RNA and total RNA synthesis also vary with the carbon source at 42 degrees C. Linear regression analysis shows only a moderately good correlation (correlation coefficient = 0.62, P = 0.0001) between the ppGpp level at 42 degrees C and the rate of rRNA synthesis at 42 degrees C. In fact, ppGpp levels are a slightly better predictor of the rate of total RNA synthesis (correlation coefficient = 0.69, P = 0.0001) at 42 degrees C. Other variables such as rate of carbon source uptake appear to have very little, if any, relationship to the rate of rRNA synthesis on the different carbon sources. Segmented linear regression analysis indicates that ppGpp levels and rates of RNA synthesis correlate best when the carbon sources are divided into two groups: 6- and 12-carbon sugars and other carbon sources. The rate of rRNA synthesis in 2S142 at 42 degrees C appears to be relatively insensitive to ppGpp levels with 6- and 12-carbon sugars as the carbon source. These data raise the possibility that carbon source may affect rRNA synthesis in a manner that is at least partially unrelated to ppGpp levels.     

Effect of guanosine tetraphosphate on in vitro protein synthesis directed by E1 and E3 colicinogenic factors.

The in vitro synthesis of colicin E3 was found to be stimulated by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), while that of several other ColE3 plasmid-specific proteins was reduced in the presence of this nucleotide. The ColE1 plasmid-directed in vitro synthesis of colicin E1 was also found to be stimulated by ppGpp.     

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3''(2'')-diphosphate 5''-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

Unusual effects of 5a,6-anhydrotetracycline and other tetracyclines. Inhibition of guanosine 5'-diphosphate 3'-diphosphate metabolism, RNA accumulation and other growth-related processes in Escherichia coli.

5a,6-Anhydrotetracycline was discovered to be unique among several tetracycline derivatives tested in its ability to inhibit RNA accumulation in vivo at low concentration (20 microgram/ml and less). In addition, in vivo protein, DNA, and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthesis were completely inhibited by 20 microgram/ml 5a,6-anhydrotetracycline. ppGpp decay in a spoT strain was inhibited by 20 microgram/ml 5a,6-anhydrotef RNA synthesis by a 5a,6-anhydrotetracycline may be due, in part, to reduced UTP and CTP synthesis. The effects of tetracyclines on in vitro ppGpp synthesis by crude stringent factor in the absence of ribosomes were investigated. It was determined that of six tetracyclines tested, four strongly inhibited the reaction (oxytetracycline, chlorotetracycline, dedimethylaminotetracycline, and tetracycline) whereas 5a,6-anhydrotetracycline gave a moderate inhibition and alpha-6-deoxyoxytetracycline resulted in only a slight reduction in ppGpp synthesis. It is proposed that tetracyclines interfere with factors involved in ppGpp metabolism and function.     

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.     

Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956.

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.     

Escherichia coli mutant containing a large deletion from relA to argA.

A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Nitrogenase synthesis in Klebsiella pneumoniae: enhanced nif expression without accumulation of guanosine 5'-diphosphate 3'-diphosphate

Derepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+4-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 micrograms ml-1), expression from the nifH and nifL promoters, determined as beta-galactosidase activity in nif::lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.     

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.     

Carbon source transport, nucleotide levels, and stable RNA synthesis in a mutant strain of Escherichia coli

We have previously described a temperature-sensitive mutant of Escherichia coli, 2S142 (rel-, met-, rns-, ilv-, ts-) which shows specific inhibition of stable RNA synthesis at 42 degrees C. This mutation mimics a carbon source downshift in that the decay of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) is inhibited at the restrictive temperature. In this paper we show that the temperature-sensitive lesion in 2S142 does affect the uptake of glucose or alpha-D-methylglucopyranoside (alpha DMG) at 42 degrees C. However, restoration of glucose or alpha DMG uptake by the insertion of a constitutive galactose permease gene or further restriction of glucose uptake by insertion of a ptsG mutation into 2S142 have no effect on rRNA synthesis at 42 degrees C (although ppGpp levels are lowered in both cases). Furthermore, while restriction of uptake at 42 degrees C varies widely from carbon source to carbon source, severe restriction of rRNA synthesis is observed on all carbon sources tested at 42 degrees C. Levels of glycolytic intermediates, adenylate energy charge, ATP levels, and cAMP levels are all unaffected at the restrictive temperature. GTP levels decrease at 42 degrees C in glucose grown cells but that also does not appear to be related to the decrease in rRNA synthesis. These data were interpreted to suggest that the restriction of stable RNA synthesis in 2S142 at 42 degrees C can not be explained on the basis of decreased uptake and/or metabolism of carbon source. "Phantom spot" levels do decrease in 2S142 at 42 degrees C. In fact, "phantom spot" is the only putative regulatory molecule which correlates with restriction of rRNA synthesis on all carbon sources tested.     

Selective inhibition of tRNATyr transcription by guanosine 3'-diphosphate 5'-diphosphate.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) selectively reduces the synthesis of su+III tRNA from omega 80 psu+III DNA relative to the synthesis of omega 80 RNA in a system in vitro containing DNA and Escherichia coli RNA polymerase holoenzyme as the sole macromolecular components. The response of su+III tRNA synthesis to increasing salt and to temperature in the presence of ppGpp suggests that the nucleotide may reduce the affinity of the enzyme for su+III promoters. The Ki for the selective inhibition of tRNA synthesis by ppGpp is 4 muM in contrast to the value of 150 muM for the inhibition of rRNA synthesis.     

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.     

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

Influence of amino acid starvation on guanosine 5''-diphosphate 3''-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.     

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.