RESUMEN
The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nifiscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nifiscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold.
Asunto(s)
Azotobacter vinelandii , Escherichia coli , Fijación del Nitrógeno , Nitrogenasa , Azotobacter vinelandii/genética , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Nitrogenasa/metabolismo , Nitrogenasa/genética , Fijación del Nitrógeno/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Glutamate is an essential biological compound produced for various therapeutic and nutritional applications. The current glutamate production process requires a large amount of ammonium, which is generated through the energy-consuming and CO2-emitting Haber-Bosch process; therefore, the development of bio-economical glutamate production processes is required. We herein developed a strategy for glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca. We showed that a simultaneous supply of glucose and citrate as carbon sources enhanced the nitrogenase activity of K. oxytoca. In the presence of glucose and citrate, K. oxytoca strain that was genetically engineered to increase the supply of 2-oxoglutarate, a precursor of glutamate synthesis, produced glutamate extracellularly more than 1 g L-1 from aerial nitrogen. This strategy offers a sustainable and eco-friendly manufacturing process to produce various nitrogen-containing compounds using aerial nitrogen.
Asunto(s)
Ácido Glutámico , Klebsiella oxytoca , Klebsiella oxytoca/genética , Nitrógeno , Ácido Cítrico , Ingeniería Metabólica , GlucosaRESUMEN
Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops.Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine.
Asunto(s)
Bacteriocinas/química , Colicinas/química , Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN Bacteriano/química , ARN de Transferencia de Arginina/química , Ribosomas/metabolismo , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Emparejamiento Base , Sitios de Unión , Colicinas/genética , Colicinas/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismo , Ribosomas/genética , Especificidad por Sustrato , Tiouridina/análogos & derivados , Tiouridina/metabolismo , Uridina/análogos & derivados , Uridina/metabolismoRESUMEN
Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.
Asunto(s)
Azorhizobium caulinodans/genética , Proteínas Bacterianas/genética , Eliminación de Gen , Operón , Tallos de la Planta/microbiología , Regulación Bacteriana de la Expresión Génica , Mutación , Fijación del Nitrógeno , Sesbania/microbiología , Simbiosis , Factores de Transcripción/genéticaRESUMEN
Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Medios de Cultivo/química , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Escherichia coli/genética , MutaciónRESUMEN
A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Estrés Fisiológico/genética , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Expresión Génica , Biblioteca de GenesRESUMEN
Bacteria have multiple K+ uptake systems. Escherichia coli, for example, has three types of K+ uptake systems, which include the low-K+-inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K+ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix- nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix+ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix+ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K+ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K+ conditions. The cytoplasmic K+ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdpFABC under high-K+ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K+ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K+ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K+ uptake systems to control the cytoplasmic K+ levels. In many bacteria, the K+ uptake system KdpFABC is expressed under low-K+ conditions. For years, many researchers have argued over how bacteria sense K+ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K+ concentrations, the detailed mechanism of K+ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K+ concentration and that high extracellular K+ concentrations repress the expression of KdpFABC via TrkJ.
Asunto(s)
Azorhizobium caulinodans/fisiología , Proteínas Bacterianas/genética , Fabaceae/microbiología , Regulación Bacteriana de la Expresión Génica , Potasio/metabolismo , Proteínas Represoras/metabolismo , Simbiosis , Proteínas Bacterianas/metabolismo , Fabaceae/fisiología , Operón , Filogenia , Proteínas Represoras/genéticaRESUMEN
R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.
Asunto(s)
Azorhizobium caulinodans/genética , Azorhizobium caulinodans/patogenicidad , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Cuerpos de Inclusión/metabolismo , Simbiosis , Azorhizobium caulinodans/ultraestructura , Proteínas Bacterianas/metabolismo , Frío , Fabaceae/microbiología , Eliminación de Gen , Cuerpos de Inclusión/ultraestructura , Ácidos Cetoglutáricos/farmacología , Operón , Paramecium/microbiología , Regiones Promotoras GenéticasRESUMEN
In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.
Asunto(s)
Aminoácidos/química , Péptidos/química , Hidrólisis , EstereoisomerismoRESUMEN
RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Colicinas/metabolismo , Hidrólisis , Viabilidad MicrobianaRESUMEN
Ovalbumin, a major protein in chicken egg white, is converted into a more thermostable molecular form, known as S-ovalbumin, during the storage of shell eggs. Our previous X-ray crystallographic study indicated that S-ovalbumin contains three D-Ser residues (S164, S236, and S320), which may account for its thermostability. Here, we confirmed the presence of these D-Ser residues in ovalbumin using a technique combining deuterium labeling of α-protons of amino acids and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ovalbumin from chicken egg white and recombinant ovalbumin were incubated for approximately 12 days at pH 9.5 and 37°C. They were then hydrolyzed in DCl/D2O vapor, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and analyzed by LC-MS/MS. A time-dependent increase in the D-Ser contents in native ovalbumin was observed over a period of 7 days, reaching approximately 8%. This corresponds to a value of three serine residues per molecule, and is consistent with the prediction based on our previous crystallographic analysis. Nearly identical results were obtained with recombinant ovalbumin. We then used this technique to investigate whether D-amino acid residues could arise within other proteins under mild alkaline conditions and detected small but significant amounts of D-Ala and/or D-Ser residues that increased in a time-dependent manner in some proteins.
Asunto(s)
Calor , Ovalbúmina/química , Serina/química , Animales , Bovinos , Pollos , Ovalbúmina/metabolismo , Estabilidad Proteica , Serina/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
In previous report, we detected D-amino acids in the acid hydrolysates of purified recombinant ß-galactosidase. Here, we employed a deuterium-hydrogen exchange method to discriminate innate D-amino acids from those generated during hydrolytic incubation. After hydrolysis of ß-galactosidase in DCl/D2O, amino acids were derivatized with NBD-F and separated on a reverse-phase column, followed by liquid chromatography-tandem mass spectrometry equipped with a chiral column. Our results show an absence of innate D-amino acid residues in the protein and suggest that the protein undergoes isomerization during a very early stage of hydrolytic incubation.
Asunto(s)
Aminoácidos/análisis , Proteínas de Escherichia coli/análisis , Hidrolisados de Proteína/análisis , Espectrometría de Masas en Tándem/métodos , beta-Galactosidasa/análisis , Aminoácidos/metabolismo , Cromatografía Liquida/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrólisis , Espectrometría de Masas/métodos , Hidrolisados de Proteína/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ(0) cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , ARN/metabolismo , Secuencia de Bases , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Datos de Secuencia Molecular , ARN/genética , ARN Mitocondrial , ARN de Transferencia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The killer yeast species Pichiaacaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNA(Gln)mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.
Asunto(s)
División del ADN , Factores Asesinos de Levadura/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Puntos de Control del Ciclo Celular , Núcleo Celular/metabolismo , Daño del ADN , Histonas/metabolismo , Fosforilación , Biosíntesis de Proteínas , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli. We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3'-cyclic phosphate ends suggested a similar mechanism to those of RNases A and T1, E5-CRD does not have the His residues necessary as a catalyst in usual RNases. To identify residues important for the catalytic reaction, mutants as to all residues within 5 Å from the central phosphorus of the scissile phosphodiester bond were prepared. Evaluation of the killing activities of the mutant colicins and the RNase activities of the mutant E5-CRDs suggested direct involvement of Arg33, Lys25, Gln29 and Lys60 in the reaction. Particularly, Arg33 plays a critical role and Ile94 provides a structural support of Arg33. Crystal structure of the complex of E5-CRD(R33Q)/dGpdUp showed structural and binding functional integrity of this mutant protein, suggesting involvement of Arg33 in the catalytic reaction. The structure of the free E5-CRD, we also determined, showed great flexibility of a flap region, which facilitates the access of Lys60 to the substrate in an induced-fit manner.
Asunto(s)
Biocatálisis , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Endorribonucleasas/genética , Proteínas de Escherichia coli/genética , Histidina , Modelos Moleculares , Mutagénesis Sitio-Dirigida , ARN de Transferencia/metabolismo , Ribonucleasas/metabolismoRESUMEN
Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Factores Asesinos de Levadura/farmacología , División del ARN/efectos de los fármacos , ARN de Transferencia/química , Supervivencia Celular/efectos de los fármacos , Fase G1/efectos de los fármacos , Fase G1/genética , Biosíntesis de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Transcripción GenéticaRESUMEN
The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.
Asunto(s)
Aminoácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Péptidos/química , Espectrometría de Masas en Tándem/métodos , Deuterio/química , Medición de Intercambio de Deuterio , Hidrólisis , EstereoisomerismoRESUMEN
It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.
Asunto(s)
Aminoácidos/análisis , Escherichia coli/genética , Urocortinas/química , beta-Galactosidasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , ADN de Cadena Simple/genética , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Urocortinas/genética , Urocortinas/aislamiento & purificación , beta-Galactosidasa/genética , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Colicin D is a plasmid-encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA(Arg) species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating-related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing cell growth. The pheromone-signalling pathway was activated only in alpha cells by tRNA cleavage, which was not observed in 'a' cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant cellular response is discussed.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Proliferación Celular , Citosol/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Feromonas/metabolismo , Plásmidos/genética , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia de Arginina/química , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Transcripción Genética , Transformación GenéticaRESUMEN
Colicin E5 is a ribonuclease that specifically cleaves tRNA(Tyr), tRNA(His), tRNA(Asn) and tRNA(Asp) of sensitive Escherichia coli cells by recognizing their anticodon sequences. Since all organisms possess universal anticodons of these tRNAs, colicin E5 was expected to potentially cleave eukaryotic tRNAs. Here, we expressed the active domain of colicin E5 (E5-CRD) in Saccharomyces cerevisiae and investigated its effects on growth. E5-CRD impaired growth of host cells by cleaving tRNA(Tyr), tRNA(His), tRNA(Asn) and tRNA(Asp) in S. cerevisiae, which is the same repertoire as that in E. coli. This activity of E5-CRD was inhibited by the co-expression of its cognate inhibitor (ImmE5). Notably, the growth impairment by E5-CRD was reversible; cells restored the colony-forming activity after suppression of the E5-CRD expression. This seems different from the sharp killing effect of E5-CRD on E. coli. These results may provide insights into the role and behaviour of cytosolic tRNAs on cell growth and proliferation.