RESUMEN
UNLABELLED: Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCE: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Eliminación de Gen , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Alineación de Secuencia , Streptomyces coelicolor/genéticaRESUMEN
BACKGROUND: AdpA is a key transcriptional regulator involved in the complex growth cycle of Streptomyces. Streptomyces are Gram-positive bacteria well-known for their production of secondary metabolites and antibiotics. Most work on AdpA has been in S. griseus, and little is known about the pathways it controls in other Streptomyces spp. We recently discovered interplay between ClpP peptidases and AdpA in S. lividans. Here, we report the identification of genes directly regulated by AdpA in S. lividans. RESULTS: Microarray experiments revealed that the expression of hundreds of genes was affected in a S. lividans adpA mutant during early stationary phase cultures in YEME liquid medium. We studied the expression of the S. lividans AdpA-regulated genes by quantitative real-time PCR analysis after various times of growth. In silico analysis revealed the presence of potential AdpA-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that AdpA binds directly to their promoter regions. This work identifies new pathways directly controlled by AdpA and that are involved in S. lividans development (ramR, SLI7885 also known as hyaS and SLI6586), and primary (SLI0755-SLI0754 encoding CYP105D5 and Fdx4) or secondary (cchA, cchB, and hyaS) metabolism. CONCLUSIONS: We characterised six S. lividans AdpA-dependent genes whose expression is directly activated by this pleiotropic regulator. Several of these genes are orthologous to bldA-dependent genes in S. coelicolor. Furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by S. lividans AdpA, although few have been described as being part of any Streptomyces AdpA regulons. This study increases the number of known AdpA-regulated pathways in Streptomyces spp.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Regulón , Metabolismo Secundario , Streptomyces lividans/genética , Transactivadores/metabolismo , Sitios de Unión , Biología Computacional , Medios de Cultivo/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Análisis por Micromatrices , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Streptomyces lividans/crecimiento & desarrollo , Streptomyces lividans/metabolismo , Factores de TiempoRESUMEN
Insertion of an apramycin resistance cassette in the clpP1clpP2 operon (encoding the ClpP1 and ClpP2 peptidase subunits) affects morphological and physiological differentiation of Streptomyces lividans. Another key factor controlling Streptomyces differentiation is the pleiotropic transcriptional regulator AdpA. We have identified a spontaneous missense mutation (-1 frameshift) in the adpA (bldH) open reading frame in a clpP1clpP2 mutant that led to the synthesis of a non-functional AdpA protein. Electrophoretic mobility shift assays showed that AdpA bound directly to clpP1clpP2 promoter region. Quantitative real-time PCR analysis showed that AdpA regulated the clpP1clpP2 operon expression at specific growth times. In vitro, AdpA and ClgR, a transcriptional activator of clpP1clpP2 operon and other genes, were able to bind simultaneously to clpP1 promoter, which suggests that AdpA binding to clpP1 promoter did not affect that of ClgR. This study allowed to uncover an interplay between the ClpP peptidases and AdpA in S. lividans.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Operón/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Mutación , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Transactivadores/genéticaRESUMEN
ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.
Asunto(s)
Antibacterianos/metabolismo , Depsipéptidos/metabolismo , Farmacorresistencia Bacteriana , Endopeptidasa Clp/antagonistas & inhibidores , Streptomyces lividans/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Inhibidores de Proteasas/metabolismo , Streptomyces lividans/metabolismoRESUMEN
To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) DeltadnaK strain of Escherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.
Asunto(s)
Escherichia coli/metabolismo , Eliminación de Gen , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas Recombinantes/biosíntesis , Escherichia coli/genética , Proteínas de Escherichia coliRESUMEN
It has been shown previously that expression of the Streptomyces lividans clpP1P2 operon, encoding proteolytic subunits of the Clp complex, the clpC1 gene, encoding the ATPase subunit, and the lon gene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes the clgR gene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in a clpP1P2 mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteasa La/metabolismo , Regulón , Streptomyces lividans/genética , Transactivadores/metabolismo , Proteínas Bacterianas/genética , Western Blotting , Operón , ARN Bacteriano/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptomyces lividans/enzimología , Transactivadores/genética , Transcripción GenéticaRESUMEN
ssrA genes encoding tmRNA with transfer and messenger RNA functions are ubiquitous in bacteria. In a process called trans-translation, tmRNA enters a stalled ribosome and allows release of the original mRNA, then tmRNA becomes the template for translation of a short tag that signals for proteolytic degradation. We provide here the first evidences that the tmRNA tagging system (ssrA and cohort smpB) is active in Streptomyces. Transcription of the genes was shown and construction of a genetic probe allowed detection of a tmRNA-tagged peptide. Obtention of ssrA and smpB mutants of Streptomyces lividans showed that the ssrA system is dispensable in Streptomyces. Morphologies of the mutants colonies were similar to the wild type, thus tmRNA-mediated tagging does not seem to have, under conditions used, a significant effect in the Streptomyces differentiation.
Asunto(s)
Biosíntesis de Proteínas/fisiología , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Streptomyces/fisiología , Proteínas Bacterianas/fisiología , Endopeptidasas/genética , Regulación Bacteriana de la Expresión Génica , Streptomyces/genética , Streptomyces coelicolor/fisiología , Streptomyces lividans/fisiologíaRESUMEN
The clp genes encoding the Clp proteolytic complex are widespread among living organisms. Five clpP genes are present in Streptomyces. Among them, the clpP1 clpP2 operon has been shown to be involved in the Streptomyces growth cycle, as a mutation blocked differentiation at the substrate mycelium step. Four Clp ATPases have been identified in Streptomyces coelicolor (ClpX and three ClpC proteins) which are potential partners of ClpP1 ClpP2. The clpC1 gene appears to be essential, since no mutant has yet been obtained. clpP1 clpP2 and clpC1 are important for Streptomyces growth, and a study of their regulation is reported here. The clpP3 clpP4 operon, which has been studied in Streptomyces lividans, is induced in a clpP1 mutant strain, and regulation of its expression is mediated via PopR, a transcriptional regulator. We report here studies of clgR, a paralogue of popR, in S. lividans. Gel mobility shift assays and DNase I footprinting indicate that ClgR binds not only to the clpP1 and clpC1 promoters, but also to the promoter of the Lon ATP-dependent protease gene and the clgR promoter itself. ClgR recognizes the motif GTTCGC-5N-GCG. In vivo, ClgR acts as an activator of clpC1 gene and clpP1 operon expression. Similarly to PopR, ClgR degradation might be ClpP dependent and could be mediated via recognition of the two carboxy-terminal alanine residues.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas , Serina Endopeptidasas/genética , Streptomyces/genética , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Secuencia de Bases , Endopeptidasa Clp , Datos de Secuencia Molecular , OperónRESUMEN
Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.
Asunto(s)
Actinomyces/genética , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Proteínas de Choque Térmico/metabolismo , Péptido Hidrolasas/genética , Regulón/genética , Proteínas Represoras/metabolismo , Western Blotting , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Mutación/genética , Péptido Hidrolasas/metabolismo , Proteínas Represoras/genética , Esporas Bacterianas , Streptomyces/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Five clpP genes have been identified in Streptomyces coelicolor. The clpP1 and clpP2 genes form one operon, the clpP3 and clpP4 genes form another, and clpP5 is monocistronic. Previous studies in Streptomyces lividans have shown that the first operon (clpP1 clpP2) is required for a normal cell cycle. Expression of the second operon (clpP3 clpP4) is activated by PopR if the first operon is nonfunctional. We show here that PopR degradation is primarily dependent on ClpP1 and ClpP2, but can also be achieved by ClpP3 and ClpP4. The carboxy-terminus of PopR plays an essential part in the degradation process. Indeed, replacement of the last two alanine residues by aspartate residues greatly increased PopR stability. These substitutions did not impair PopR activity and, as expected, accumulation of the mutant form of PopR led to very strong expression of the clpP3 clpP4 operon. Increased PopR levels led to delayed sporulation. The results obtained in this study support the notion of cross-processing between ClpP1 and ClpP2.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón/genética , Serina Endopeptidasas/metabolismo , Streptomyces/genética , Transactivadores/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfato/metabolismo , Alanina/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Consenso , Endopeptidasa Clp , Endopeptidasas/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Streptomyces/metabolismo , Especificidad por Sustrato , Transactivadores/genéticaRESUMEN
In Streptomyces albus, Hsp18, a protein belonging to the family of small heat-shock proteins, can be detected only at high temperature. Disruption of orfY, located upstream and in the opposite orientation to hsp18, resulted in an elevated level of hsp18 mRNA at low temperature. Genetic and biochemical experiments indicated that the product of orfY, now called RheA (Repressor of hsp eighteen), directly represses hsp18. In Escherichia coli, an hsp18'-bgaB transcriptional fusion was repressed in a strain expressing S. albus RheA. DNA-binding experiments with crude extracts of E. coli overproducing RheA indicated that RheA interacts specifically with the hsp18 promoter. Transcription analysis of rheA in the S. albus wild-type and in rheA mutant strains suggested that RheA represses transcription not only of hsp18 but also of rheA itself.