Characterization of peptide chain length and constituency requirements for YejABEF-mediated uptake of microcin C analogues

Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.     

Cloning and mapping of the genetic determinants for microcin C7 production and immunity.

Microcin C7, a peptide antibiotic inhibitor of protein synthesis, is produced by Escherichia coli K-12 strains that carry the 43-kilobase low-copy-number plasmid pMccC7. Microcin C7 production and immunity determinants of this plasmid have been cloned into the vectors pBR322 and pACYC184. The resulting plasmids overproduce microcin C7 and express immunity against the microcin. Mcc- and Mcc- Imm- mutants have been isolated on recombinant plasmids by inserting transposable elements. Physical and phenotypic characterization of these mutants shows that a DNA region of 5 kilobases is required to produce microcin C7, and that two small regions located inside the producing region are also required to express immunity. Analysis of plasmids carrying mcc-lacZ gene fusions indicates that all microcin DNA is transcribed in the same direction. The results suggest that a structure like a polycistronic operon is responsible for microcin C7 production and immunity.     

Amino acid residues required for maturation, cell uptake, and processing of translation inhibitor microcin C

Microcin C (McC), a peptide-nucleotide Trojan horse antibiotic, targets aspartyl-tRNA synthetase. We present the results of a systematic mutational study of the 7-amino-acid ribosomally synthesized peptide moiety of McC. Our results define amino acid positions important for McC maturation and cell uptake and processing and open the way for creation of more potent McC-based inhibitors.     

Fragments of the Bacterial Toxin Microcin B17 as Gyrase Poisons

Fluoroquinolones are very important drugs in the clinical antibacterial arsenal; their success is principally due to their mode of action: the stabilisation of a gyrase-DNA intermediate (the cleavage complex), which triggers a chain of events leading to cell death. Microcin B17 (MccB17) is a modified peptide bacterial toxin that acts by a similar mode of action, but is unfortunately unsuitable as a therapeutic drug. However, its structure and mechanism could inspire the design of new antibacterial compounds that are needed to circumvent the rise in bacterial resistance to current antibiotics. Here we describe the investigation of the structural features responsible for MccB17 activity and the identification of fragments of the toxin that retain the ability to stabilise the cleavage complex.     

Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.     

Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC (2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable amounts. Using this synthesis a number of diverse compounds was synthesized with altered target specificity. Further characteristics of the YejABEF transporters were determined using these compounds.     

Microcin C: biosynthesis, mode of action, and potential as a lead in antibiotics development

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.     

Microcin C: Biosynthesis,Mode of Action,and Potential as a Lead in Antibiotics Development

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.     

Upf1 and Upf2 proteins mediate normal yeast mRNA degradation when translation initiation is limited.

mRNA degradation is coupled with the process of mRNA translation. For example, an mRNA molecule, on which translation is prematurely terminated because of a nonsense codon, may be rapidly degraded. This nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae is mediated by the Upf1 and Upf2 proteins. Yeast mRNAs can also be selectively destabilized by limiting the rate of translation initiation. Two such destabilized mRNAs, from the SSA1 and SSA2 genes, have been identified using temperature-sensitive mutations affecting the Prt1 component of eukaryotic initiation factor 3. For SSA1 and SSA2 mRNAs, and for structurally modified SSA mRNA derivatives, I show here that degradation is triggered when translation initiation is limited but ongoing. This initiation-dependent mRNA degradation is limited to a subset of mRNAs that includes at least those from the SSA1 and SSA2 genes, and occurs through Upf1- and Upf2-mediated processes, although sequence elements characteristic of nonsense-mediated decay are not evident in these mRNAs.     

The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells

In poliovirus-infected HeLa cells, the mechanism of protein synthesis initiation factor recognition of m7G cap groups on mRNA is impaired. Translation of capped host cell mRNAs is inhibited, whereas translation of uncapped poliovirus mRNA proceeds exclusively. The site of this defect has been localized to the cap-binding protein complex (CBPC). To elucidate the specific structural and functional defects of the CBPC following poliovirus infection, the CBPC and/or its polypeptide components were purified from uninfected and poliovirus-infected HeLa cells. The CBPC from uninfected cells consisted of tightly associated 24- and 220-kDa polypeptides; minor amounts of polypeptides of 40, 44, and 80 kDa also consistently co-purified with the p24/p220 cores. No evidence of a 50-kDa, eIF-4A-related polypeptide subunit of the CBPC was obtained. The CBPC from poliovirus-infected cells had undergone major structural alterations. The 220-kDa component was absent; antigenically related (100-130 kDa) degradation products were present instead. The 24-kDa component co-purified with the p220 degradation products, but other components were missing. The association of the infected cell CBPC components was quite labile compared with that demonstrated by the components of CBPC from uninfected cells. Differential stimulation of capped, but not uncapped mRNAs in a cell-free translation assay was demonstrated by unmodified CBPC. Conversely, modified CBPC from poliovirus-infected cells differentially stimulated in vitro translation of uncapped poliovirus mRNA but not capped mRNAs. The implications of these results for the mechanism of cap-independent translation are briefly discussed.     

Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites

The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.     

Microcin 7: purification and properties

Microcin 7 is an antibiotic peptide, produced and excreted to the culture medium by E. coli strains harboring the plasmid pRYC7. This peptide was extracted from the culture media by adsorbing it on octadecyl silica. It was purified by gel filtration on Sephadex G-25 and reverse phase high performance liquid chromatography. Its amino acid composition is the following: Ala (0.8), Arg (1.9), Asx (1.9), Gly (1.5), Met (0.8) and Thr (0.9). The purified peptide dose not react with ninhydrin and it is resistant to carboxypeptidase degradation, indicating that the molecule may be a cyclic or end-blocked oligopeptide.     

sbmC, a stationary-phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17

Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC , that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids ( M_r , 18 095) that has been visualized in SDS—polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G → T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.     

A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis

Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.     

Elongation factor 2 diphthamide is critical for translation of two IRES-dependent protein targets,XIAP and FGF2, under oxidative stress conditions

Elongation factor-2 (eEF2) catalyzes the movement of the ribosome along the mRNA. A single histidine residue in eEF2 (H715) is modified to form diphthamide. A role for eEF2 in the cellular stress response is highlighted by the fact that eEF2 is sensitive to oxidative stress and that it must be active to drive the synthesis of proteins that help cells to mitigate the adverse effects of oxidative stress. Many of these proteins are encoded by mRNAs containing a sequence called an “internal ribosomal entry site” (IRES). Under high oxidative stress conditions diphthamide-deficient cells were significantly more sensitive to cell death. These results suggest that diphthamide may play a role in protection against the degradation of eEF2. This protection is especially important in those situations in which eEF2 is necessary for the reprogramming of translation from global to IRES synthesis. Indeed, we found that the expression of X-linked inhibitor of apoptosis (XIAP) and fibroblast growth factor 2 (FGF2), two proteins synthesized from mRNAs with IRESs that promote cell survival, is deregulated in diphthamide-deficient cells. Our findings therefore suggest that eEF2 diphthamide controls the selective translation of IRES-dependent protein targets XIAP and FGF2, critical for cell survival under conditions of oxidative stress.     

N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids

Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.     

Scd6 targets eIF4G to repress translation: RGG motif proteins as a class of eIF4G-binding proteins

The formation of mRNPs controls the interaction of the translation and degradation machinery with individual mRNAs. The yeast Scd6 protein and its orthologs regulate translation and mRNA degradation in yeast, C.?elegans, D.?melanogaster, and humans by an unknown mechanism. We demonstrate that Scd6 represses translation by binding the eIF4G subunit of eIF4F in a manner dependent on its RGG domain, thereby forming an mRNP repressed for translation initiation. Strikingly, several other RGG domain-containing proteins in yeast copurify with eIF4E/G and we demonstrate that two such proteins, Npl3 and Sbp1, also directly bind eIF4G and repress translation in a manner dependent on their RGG motifs. These observations identify the mechanism of Scd6 function through its RGG motif and indicate that eIF4G plays an important role as a scaffolding protein for the recruitment of translation repressors.     

The DNA replication inhibitor microcin B17 is a forty-three-amino-acid protein containing sixty percent glycine

Microcin B17 is a low-molecular-weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70-kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high-performance liquid chromatography through a C18 column. The N-terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N-terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N-terminal peptide does not have the characteristic properties of a "signal sequence", which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.