The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.     

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.     

Male-Specific Bacteriophage MS2 Propagation in Fluorophenylalanine-Resistant Escherichia coli K12

Mutation of Escherichia coli K12 HfrH to resistance to fluorophenylalanine resulted in changes in the plaque morphology of bacteriophage MS2 on this strain and led to an increased efficiency of propagation of the phage in liquid cultures. Evidence was obtained that the mutation resulted in inhibition of early lysis in infected cells and that lysis involved the production of a lysozyme. Genetic studies suggested that the observed pleiotropy of the resistance mutation was due to informational suppression.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.     

Rifampin-resistant RNA polymerase in spirochetes

Abstract Various free-living and host-associated spirochetes isolated by methods not involving rifampin were resistant to relatively high concentrations of this antibiotic. The lowest concentrations of rifampin that were inhibitory for the spirochetes ranged from 50 to more than 200 μ g/ml, depending on the species. The spirochete strains examined were at least 10-fold more resistant to rifampin than Escherichia coli and 10 000-fold more resistant than Staphylococcus aureus . The results support the conclusion that rifampin resistance is a general characteristic of spirochetes. Resistance of Spirochaeta aurantia to rifampin was not the result of detoxification of the antibiotic in the culture medium. The activity of spirochete DNA-dependent RNA polymerase in vitro was completely resistant to 10 μg of rifampin per ml, a concentration that totally inhibited E. coli RNA polymerase. Higher concentrations decreased the spirochetal activity. Thus, rifampin resistance may be due to a low affinity of spirochete RNA polymerase for the antibiotic.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).     

Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor.

Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

Inhibition of growth of cells in culture by L-phenylalanine as a model system for the analysis of phenylketonuria. I. Amino acid antagonism and the inhibition of protein synthesis

Phenylalanine in high concentrations inhibits the growth of mouse A9 cells. Protein synthesis is inhibited earlier and more severely than RNA or DNA synthesis. Phenylalanine inhibits the uptake and decreases the intracellular pool of several amino acids. Certain amino acids added in excess reverse the phenylalanine inhibition. The strongest reversing amino acids appear to function by excluding phenylalanine. The phenylalanine inhibition does not appear to be due to a deficiency of any amino acid, but to the high intracellular phenylalanine concentration and/or an amino acid imbalance resulting from the large ratio of phenylalanine to other amino acids.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by `magic spot'' nucleotides

During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.     

Regulation of RNA replication in RNA-containing bacteriphages. RNA synthesis in coat protein polar mutants]

The synthesis of RNA by polar coat protein mutants f2sus3 and Qbetaam12 under suppressor (Escherichia coli S26R1E, Su+-1; H12R8a Su+-3) and non-suppressor (E. coli AB259; S26) conditions was examined. It was demonstrated that the synthesis of viral RNA under non-suppressor conditions in the presence of rifamycin produced the same gaussian pattern of rates as the synthesis of RNA by wild type phage or non-polar coat protein mutants. However, the total amount of RNA was decreased approximately 10-fold and the peak of RNA synthesis was displaced 7--10 min later. The number of infective centers was reduced also 10-fold indicating that a certain time-lapse was required to overcome the polarity of the parental RNA, this process being of single occurrence, exclusively on the parental RNA, but not on the progeny strains. As a consequence, it was concluded that the initiation of translation at the replicase cistron starts on the nascent RNA chains within the replicative complexes and not on the fully-synthesized templates with their complete secondary structure. The data obtained are not in contradiction with the hypothesis concerning the role of the repressor complex II (replicase-RNA) to slow down the synthesis of replicase and RNA in the coat protein mutants. The polarity can not be responsible probably for the blocking of the replicase cistron on the nascent chain following the block of coat protein cistron. Therefore, it appears appropriate to assume the existence of two binding sites for the replicase as repressor which is in keeping with the conclusions of Weissmann and co-workers.     

Development of a microRNA delivery system based on bacteriophage MS2 virus-like particles

Recently, microRNA (miRNA)-mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA-mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for RNA and drug delivery. However, MS2 VLP-mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre-miR 146a contained MS2 VLPs, and then conjugated these particles with HIV-1 Tat(47-57) peptide. The conjugated MS2 VLPs effectively transferred the packaged pre-miR146a RNA into various cells and tissues, with 0.92-14.76-fold higher expression of miR-146a in vitro and about two-fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Biochemical and Autoradiographic Study of Cerebral Protein Synthesis with [18F]- and [14C]Fluorophenylalanine

Fluorine-18-labeled ortho or para isomers of L-fluorophenylalanine were used in double-label experiments together with L-[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that L-p-[18F]fluorophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, L-p-fluorophenyl[3-14C]alanine and L-o-[18F]fluorophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of L-p-[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

Addressed enzymatic fragmentation of RNA molecules

RNase H has been used for selective cleavage of RNA of MS2 and R17 bacteriophages and 16S RNA from E. coli ribosomes in the region of formation of heteroduplex composed of RNA and an oligodeoxyribonucleotide complementary to a certain part of it. The oligonucleotides used--d(C-T-C-A-T-G-T-T-), d(C-C-A-T-C-T-T-T-T) and d(T-T-T-C-C-A-T-C-T-T-T-T)--were synthesized by chemical methods. The molecular weight of the fragments produced on cleavage of the RNA of MS2 and R17 were estimated with the use of gel electrophoresis under denaturating conditions. The dependence of the enzyme activity on Mg2+ and Na+ concentration and of RNA cleavage on the RNA: oligodeoxyribonucleotide ratio was investigated.     

Mutagenesis by normal metabolites in Escherichia coli: phenylalanine mutagenesis is dependent on error-prone DNA repair

In search of a model for the production of 'spontaneous' mutations induced by DNA damage produced during normal metabolism, 19 amino acids were tested for mutagenicity in Escherichia coli K-12 uvrB. Cystine, and, to a lesser extent, arginine and threonine were found to be antimutagenic; only phenylalanine was found to be mutagenic. At 2 mM, phenylalanine induced mutants at 1.5-2-fold above background [lacZ53(amber)----Lac+, rifampicin resistance (missense), and bacteriophage T6 resistance]. Tyrosine and, to a lesser extent, tryptophan (each at 2 mM) inhibited the mutagenicity of phenylalanine. Phenylalanine mutagenesis was detected in the uvrB strain, but not in the wild-type, uvrB umuC or uvrB lexA strains. Thus, phenylalanine seems to cause the production of excisable lesions ('UV-like'?) in DNA, which, if not excised, can induce mutations via error-prone DNA repair.