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1.
Cell Genom ; 3(11): 100418, 2023 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-38020971

RESUMEN

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

2.
Front Microbiol ; 12: 641756, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33717038

RESUMEN

Dalbavancin, vancomycin and chlorobiphenyl-vancomycin share a high degree of structural similarity and the same primary mode of drug action. All inhibit bacterial cell wall biosynthesis through complexation with intermediates in peptidoglycan biosynthesis mediated via interaction with peptidyl-d-alanyl-d-alanine (d-Ala-d-Ala) residues present at the termini of the intermediates. VanB-type glycopeptide resistance in bacteria encodes an inducible reprogramming of bacterial cell wall biosynthesis that generates precursors terminating with d-alanyl-d-lactate (d-Ala-d-Lac). This system in Streptomyces coelicolor confers protection against the natural product vancomycin but not dalbavancin or chlorobiphenyl-vancomycin, which are semi-synthetic derivatives and fail to sufficiently activate the inducible VanB-type sensory response. We used transcriptome profiling by RNAseq to identify the gene expression signatures elucidated in S. coelicolor in response to the three different glycopeptide compounds. An integrated comparison of the results defines both the contribution of the VanB resistance system to the control of changes in gene transcription and the impact at the transcriptional level of the structural diversity present in the glycopeptide antibiotics used. Dalbavancin induces markedly more extensive changes in the expression of genes required for transport processes, RNA methylation, haem biosynthesis and the biosynthesis of the amino acids arginine and glutamine. Chlorobiphenyl-vancomycin exhibits specific effects on tryptophan and calcium-dependent antibiotic biosynthesis and has a stronger repressive effect on translation. Vancomycin predictably has a uniquely strong effect on the genes controlled by the VanB resistance system and also impacts metal ion homeostasis and leucine biosynthesis. Leaderless gene transcription is disfavoured in the core transcriptional up- and down-regulation taking place in response to all the glycopeptide antibiotics, while HrdB-dependent transcripts are favoured in the down-regulated group. This study illustrates the biological impact of peripheral changes to glycopeptide antibiotic structure and could inform the design of future semi-synthetic glycopeptide derivatives.

3.
Curr Genet ; 65(4): 893-897, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-30937517

RESUMEN

This mini-review considers the idea that guanylate nucleotide energy charge acts as an integrative signal for the regulation of gene expression in eukaryotic cells and discusses possible routes for that signal's transduction. Gene expression is intimately linked with cell nutrition and diverse signaling systems serve to coordinate the synthesis of proteins required for growth and proliferation with the prevailing cellular nutritional status. Using short pathways for the inducible and futile consumption of ATP or GTP in engineered cells of Saccharomyces cerevisiae, we have recently shown that GTP levels can also play a role in determining how genes act to respond to changes in cellular energy supply. This review aims to interpret the importance of GTP as an integrative signal in the context of an increasing body of evidence indicating the spatio-temporal complexity of cellular de novo purine nucleotide biosynthesis.


Asunto(s)
Metabolismo Energético/genética , Nucleótidos de Guanina/genética , Transcripción Genética , Purinas/metabolismo , Saccharomyces cerevisiae/genética , Transducción de Señal/genética
4.
mBio ; 10(1)2019 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-30670615

RESUMEN

Correlations between gene transcription and the abundance of high-energy purine nucleotides in Saccharomyces cerevisiae have often been noted. However, there has been no systematic investigation of this phenomenon in the absence of confounding factors such as nutrient status and growth rate, and there is little hard evidence for a causal relationship. Whether transcription is fundamentally responsive to prevailing cellular energetic conditions via sensing of intracellular purine nucleotides, independently of specific nutrition, remains an important question. The controlled nutritional environment of chemostat culture revealed a strong correlation between ATP and GTP abundance and the transcription of genes required for growth. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into S. cerevisiae, permitting analysis of the transcriptional effect of an increased demand for these nucleotides. During steady-state growth using the fermentable carbon source glucose, the futile consumption of ATP led to a decrease in intracellular ATP concentration but an increase in GTP and the guanylate energy charge (GEC). Expression of transcripts encoding proteins involved in ribosome biogenesis, and those controlled by promoters subject to SWI/SNF-dependent chromatin remodelling, was correlated with these nucleotide pool changes. Similar nucleotide abundance changes were observed using a nonfermentable carbon source, but an effect on the growth-associated transcriptional programme was absent. Induction of the GTP-cycling pathway had only marginal effects on nucleotide abundance and gene transcription. The transcriptional response of respiring cells to glucose was dampened in chemostats induced for ATP cycling, but not GTP cycling, and this was primarily associated with altered adenine nucleotide levels.IMPORTANCE This paper investigates whether, independently of the supply of any specific nutrient, gene transcription responds to the energy status of the cell by monitoring ATP and GTP levels. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into the yeast Saccharomyces cerevisiae, and the effect of an increased demand for these purine nucleotides on gene transcription was analyzed. The resulting changes in transcription were most consistently associated with changes in GTP and GEC levels, although the reprogramming in gene expression during glucose repression is sensitive to adenine nucleotide levels. The results show that GTP levels play a central role in determining how genes act to respond to changes in energy supply and that any comprehensive understanding of the control of eukaryotic gene expression requires the elucidation of how changes in guanine nucleotide abundance are sensed and transduced to alter the global pattern of transcription.


Asunto(s)
Citosol/química , Expresión Génica , Guanosina Trifosfato/análisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo
5.
mBio ; 8(4)2017 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-28743817

RESUMEN

We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected cells recover. Despite the importance of this process, the broader impact of bacterial nucleotides on the functioning of eukaryotic cells remains poorly defined. To address this, we genetically modified cells of the eukaryote Saccharomyces cerevisiae (baker's yeast) to produce three of these molecules (cdiAMP, cdiGMP, and ppGpp) and used the engineered strains as model systems to characterize the effects of the molecules on the cells. In addition to demonstrating that the nucleotides are each capable of adversely affecting yeast cell function and growth, we also identified the cellular functions important for mitigating the damage caused, suggesting possible modes of action. This study expands our understanding of the molecular interactions that can take place between bacterial and eukaryotic cells.


Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Guanosina Difosfato/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Transducción de Señal , Ciclo Celular , Ensamble y Desensamble de Cromatina/genética , GMP Cíclico/metabolismo , Epistasis Genética , Fermentación , Perfilación de la Expresión Génica , Genes Sintéticos , Ingeniería Genética , Interacciones Huésped-Patógeno , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Saccharomyces cerevisiae/fisiología
6.
Methods Mol Biol ; 1440: 31-46, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27311662

RESUMEN

Transcriptomics, the genome-wide analysis of gene transcription, has become an important tool for characterizing and understanding the signal transduction networks operating in bacteria. Here we describe a protocol for quantifying and interpreting changes in the transcriptome of Streptomyces coelicolor that take place in response to treatment with three antibiotics active against different stages of peptidoglycan biosynthesis. The results defined the transcriptional responses associated with cell envelope homeostasis including a generalized response to all three antibiotics involving activation of transcription of the cell envelope stress sigma factor σ(E), together with elements of the stringent response, and of the heat, osmotic, and oxidative stress regulons. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. The principles behind the protocol are transferable to the study of cell envelope homeostatic mechanisms probed using alternative chemical/environmental insults or in other bacterial strains.


Asunto(s)
Proteínas Bacterianas/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Streptomyces coelicolor/genética , Antibacterianos/farmacología , Pared Celular/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Homeostasis , Calor , Estrés Oxidativo , Peptidoglicano/biosíntesis , Streptomyces coelicolor/efectos de los fármacos
7.
J Proteome Res ; 14(7): 2915-28, 2015 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-25965010

RESUMEN

Understanding how bacteria survive periods of cell wall stress is of fundamental interest and can help generate ideas for improved antibacterial treatments. In this study we use tandem mass tagging to characterize the proteomic response of vancomycin resistant Streptomyces coelicolor to the exposure to sublethal levels of the antibiotic. A common set of 804 proteins were identified in triplicate experiments. Contrasting changes in the abundance of proteins closely associated with the cytoplasmic membrane with those taking place in the cytosol identified aspects of protein spatial localization that are associated with the response to vancomycin. Enzymes for peptidoglycan precursor, mycothiol, ectoine and menaquinone biosynthesis together with a multisubunit nitrate reductase were recruited to the membrane following vancomycin treatment. Many proteins with regulatory functions (including sensor protein kinases) also exhibited significant changes in abundance exclusively in the membrane-associated protein fraction. Several enzymes predicted to be involved in extracellular peptidoglycan crossbridge formation became significantly depleted from the membrane. A comparison with data previously acquired on the changes in gene transcription following vancomycin treatment identified a common high-confidence set of changes in gene expression. Generalized changes in protein abundance indicate roles for proteolysis, the pentose phosphate pathway and a reorganization of amino acid biosynthesis in the stress response.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Proteómica , Streptomyces coelicolor/efectos de los fármacos , Estrés Fisiológico , Espectrometría de Masas en Tándem/métodos , Vancomicina/farmacología , Cromatografía Liquida , Streptomyces coelicolor/metabolismo
8.
BMC Genomics ; 12: 226, 2011 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-21569315

RESUMEN

BACKGROUND: A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2) as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin) that target distinct stages of cell wall biosynthesis. RESULTS: A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σ(E), together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σ(B) or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. CONCLUSIONS: Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional.


Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Genoma Bacteriano/genética , Streptomyces coelicolor/efectos de los fármacos , Streptomyces coelicolor/genética , Transportadoras de Casetes de Unión a ATP/genética , Biotina/biosíntesis , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Respuesta al Choque Térmico/efectos de los fármacos , Respuesta al Choque Térmico/genética , Ligasas/biosíntesis , Presión Osmótica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Peptidoglicano/biosíntesis , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , Regulón/genética , Factor sigma/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismo , Transcripción Genética/efectos de los fármacos , Zinc/farmacología
9.
Mol Biosyst ; 7(1): 139-49, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20963216

RESUMEN

Saccharomyces cerevisiae can survive extreme desiccation, but the molecular mechanisms are poorly understood. To define genes involved in desiccation tolerance, two complementary genome-wide approaches, phenomics and transcriptomics, have been used, together with a targeted analysis of gene deletion mutants tested individually for their ability to survive drying. Genome-wide phenotypic analyses carried out on a pooled library of single-gene deletion mutants subjected to three cycles of desiccation and re-growth to post-diauxic phase identified about 650 genes that contributed to strain survival in the drying process. Air-drying desiccation-tolerant post-diauxic phase cells significantly altered transcription in 12% of the yeast genome, activating expression of over 450 genes and down-regulating 330. Autophagy processes were significantly over-represented in both the phenomics study and the genes up-regulated on drying, indicating the importance of the clearance of protein aggregates/damaged organelles and the recycling of nutrients for the survival of desiccation in yeast. Functional carbon source sensing networks governed by the PKA, Tor and Snf1 protein kinase complexes were important for the survival of desiccation, as indicated by phenomics, transcriptomics, and individual analyses of mutant strains. Changes in nitrogen metabolism were evident during the drying process and parts of the environmental stress response were activated, repressing ribosome production and inducing genes for coping with oxidative and osmotic stress.


Asunto(s)
Autofagia/fisiología , Desecación , Perfilación de la Expresión Génica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Autofagia/genética , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos
10.
Mol Microbiol ; 74(6): 1427-44, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19906184

RESUMEN

The availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non-ribosomally synthesized peptide with predicted siderophore-like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc-responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor.


Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Oxazoles/metabolismo , Proteínas Represoras/fisiología , Streptomyces coelicolor/fisiología , Tiazoles/metabolismo , Zinc/metabolismo , Antraquinonas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Modelos Biológicos , Datos de Secuencia Molecular , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética
11.
Mol Microbiol ; 70(5): 1180-93, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18976279

RESUMEN

Streptomyces coelicolor produces an extracellular protease inhibitor protein, STI (Streptomyces trypsin inhibitor). We show that post-growth elimination of STI is needed for colonies to develop aerial mycelium efficiently. Inactivation of STI, and thus the normal progression of colony development, at least partly involves an extracellular protease specified by gene SCO5913. Two-hybrid analysis identified two possible targets of STI inhibition (the products of SCO1355 and SCO5447), both extracellular proteases containing a domain homologous with the P-domain of eukaryotic convertases, proteases that mediate the processing of many precursors with important cellular or developmental roles. At least the SCO1355 protease is needed for the normal progression of development. Two components of the proposed cascade are dependent on the tRNA for the rare UUA (leucine) codon, which is specified by the developmental gene bldA. A model is presented that links intracellular regulatory events with an extracellular protease cascade to facilitate normal development.


Asunto(s)
Proteínas Bacterianas/metabolismo , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Streptomyces coelicolor/crecimiento & desarrollo , Proteínas Bacterianas/genética , ADN Complementario/genética , Fermentación , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Mutación , Procesamiento Proteico-Postraduccional , ARN Bacteriano/genética , ARN de Transferencia de Leucina/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Técnicas del Sistema de Dos Híbridos
12.
BMC Genomics ; 8: 261, 2007 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-17678549

RESUMEN

BACKGROUND: In Streptomyces coelicolor, bldA encodes the only tRNA for a rare leucine codon, UUA. This tRNA is unnecessary for growth, but is required for some aspects of secondary metabolism and morphological development. We describe a transcriptomic and proteomic analysis of the effects of deleting bldA on cellular processes during submerged culture: conditions relevant to the industrial production of antibiotics. RESULTS: At the end of rapid growth, a co-ordinated transient up-regulation of about 100 genes, including many for ribosomal proteins, was seen in the parent strain but not the DeltabldA mutant. Increased basal levels of the signal molecule ppGpp in the mutant strain may be responsible for this difference. Transcripts or proteins from a further 147 genes classified as bldA-influenced were mostly expressed late in culture in the wild-type, though others were significantly transcribed during exponential growth. Some were involved in the biosynthesis of seven secondary metabolites; and some have probable roles in reorganising metabolism after rapid growth. Many of the 147 genes were "function unknown", and may represent unknown aspects of Streptomyces biology. Only two of the 147 genes contain a TTA codon, but some effects of bldA could be traced to TTA codons in regulatory genes or polycistronic operons. Several proteins were affected post-translationally by the bldA deletion. There was a statistically significant but weak positive global correlation between transcript and corresponding protein levels. Different technical limitations of the two approaches were a major cause of discrepancies in the results obtained with them. CONCLUSION: Although deletion of bldA has very conspicuous effects on the gross phenotype, the bldA molecular phenotype revealed by the "dualomic" approach has shown that only about 2% of the genome is affected; but this includes many previously unknown effects at a variety of different levels, including post-translational changes in proteins and global cellular physiology.


Asunto(s)
Medios de Cultivo/análisis , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteómica , ARN Bacteriano/genética , ARN de Transferencia de Leucina/genética , Streptomyces coelicolor/genética , Secuencia de Bases , Análisis por Conglomerados , Eliminación de Gen , Genes Bacterianos , Nucleótidos de Guanina/análisis , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Organismos Modificados Genéticamente , Procesamiento Proteico-Postraduccional/genética , Proteoma/análisis , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/metabolismo
13.
Microbiology (Reading) ; 151(Pt 8): 2707-2720, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16079348

RESUMEN

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteoma/análisis , ARN Bacteriano/metabolismo , ARN de Transferencia de Leucina/genética , Streptomyces coelicolor/metabolismo , Secuencia de Bases , ADN Bacteriano/análisis , Regulación Bacteriana de la Expresión Génica , Proteoma/genética , ARN Bacteriano/genética , ARN de Transferencia de Leucina/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrollo
14.
J Bacteriol ; 187(9): 2957-66, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15838021

RESUMEN

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.


Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Proteoma/genética , ARN Bacteriano/genética , ARN de Transferencia/genética , Streptomyces coelicolor/genética , Transactivadores/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , ADN Bacteriano , Electroforesis en Gel Bidimensional , Eliminación de Gen , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteoma/análisis , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/metabolismo , Tripsina/metabolismo
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