RESUMO
Gram-positive Streptomyces bacteria are profuse secretors of polypeptides using complex, yet unknown mechanisms. Many of their secretory proteins are proteases that play important roles in the acquisition of amino acids from the environment. Other proteases regulate cellular proteostasis. To begin dissecting the possible role of proteases in Streptomyces secretion, we applied a multi-omics approach. We probed the role of the 190 proteases of Streptomyces lividans strain TK24 in protein secretion in defined media at different stages of growth. Transcriptomics analysis revealed transcripts for 93% of these proteases and identified that 41 of them showed high abundance. Proteomics analysis identified 57 membrane-embedded or secreted proteases with variations in their abundance. We focused on 17 of these proteases and putative inhibitors and generated strains deleted of their genes. These were characterized in terms of their fitness, transcriptome and secretome changes. In addition, we performed a targeted analysis in deletion strains that also carried a secretion competent mRFP. One strain, carrying a deletion of the gene for the regulatory protease FtsH, showed significant global changes in overall transcription and enhanced secretome and secreted mRFP levels. These data provide a first multi-omics effort to characterize the complex regulatory mechanisms of protein secretion in Streptomyces lividans and lay the foundations for future rational manipulation of this process.
RESUMO
BACKGROUND: Members of the genus Streptomyces are Gram-positive bacteria that are used as important cell factories to produce secondary metabolites and secrete heterologous proteins. They possess some of the largest bacterial genomes and thus proteomes. Understanding their complex proteomes and metabolic regulation will improve any genetic engineering approach. RESULTS: Here, we performed a comprehensive annotation of the subcellular localization of the proteome of Streptomyces lividans TK24 and developed the Subcellular Topology of Polypeptides in Streptomyces database (SToPSdb) to make this information widely accessible. We first introduced a uniform, improved nomenclature that re-annotated the names of ~ 4000 proteins based on functional and structural information. Then protein localization was assigned de novo using prediction tools and edited by manual curation for 7494 proteins, including information for 183 proteins that resulted from a recent genome re-annotation and are not available in current databases. The S. lividans proteome was also linked with those of other model bacterial strains including Streptomyces coelicolor A3(2) and Escherichia coli K-12, based on protein homology, and can be accessed through an open web interface. Finally, experimental data derived from proteomics experiments have been incorporated and provide validation for protein existence or topology for 579 proteins. Proteomics also reveals proteins released from vesicles that bleb off the membrane. All export systems known in S. lividans are also presented and exported proteins assigned export routes, where known. CONCLUSIONS: SToPSdb provides an updated and comprehensive protein localization annotation resource for S. lividans and other streptomycetes. It forms the basis for future linking to databases containing experimental data of proteomics, genomics and metabolomics studies for this organism.
Assuntos
Peptídeos/metabolismo , Proteômica/métodos , Streptomyces/genéticaRESUMO
BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.
Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Expressão Gênica , Rhodothermus/enzimologia , Streptomyces lividans/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Celulase/química , Celulase/genética , Estabilidade Enzimática , Temperatura Alta , Transporte Proteico , Rhodothermus/genética , Streptomyces lividans/metabolismoRESUMO
(13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi-objective design should stimulate its application within the field of (13)C-based metabolic flux analysis.
Assuntos
Isótopos de Carbono , Análise do Fluxo Metabólico/métodos , Projetos de Pesquisa , Linhagem Celular Tumoral , Humanos , Streptomyces lividansRESUMO
The rapid rise of antibiotic-resistant bacteria is one of the major concerns in modern medicine. Therefore, to treat bacterial infections, there is an urgent need for new antibacterials-preferably directed against alternative bacterial targets. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec secretion pathway, the major route for bacterial protein export across or into the cytoplasmic membrane. As SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target. Using an in silico approach, we have defined several potentially druggable and conserved pockets on the surface of SecA. We show that three of these potentially druggable sites are important for SecA function. A starting collection of ~500 000 commercially available small-molecules was virtually screened against a predicted druggable pocket in the preprotein-binding domain of Escherichia coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of E. coli SecA. Five inhibitors of the translocation ATPase, and not of basal or membrane ATPase, were identified with IC50 values <65 µm. The most potent inhibitor showed an IC50 of 24 µm. The antimicrobial activity was determined for the five most potent SecA inhibitors. Two compounds were found to possess weak antibacterial activity (IC50 ~198 µm) against E. coli, whereas some compounds showed moderate antibacterial activity (IC50 ~100 µm) against Staphylococcus aureus.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Ligantes , Proteínas de Membrana Transportadoras , Canais de Translocação SEC , Proteínas SecARESUMO
Finding new, effective antibiotics is a challenging research area driven by novel approaches required to tackle unconventional targets. In this review we focus on the bacterial protein secretion pathway as a target for eliminating or disarming pathogens. We discuss the latest developments in targeting the Sec-pathway for novel antibiotics focusing on two key components: SecA, the ATP-driven motor protein responsible for driving preproteins across the cytoplasmic membrane and the Type I signal peptidase that is responsible for the removal of the signal peptide allowing the release of the mature protein from the membrane. We take a bird's-eye view of other potential targets in the Sec-pathway as well as other Sec-dependent or Sec-independent protein secretion pathways as targets for the development of novel antibiotics. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Assuntos
Adenosina Trifosfatases/metabolismo , Antibacterianos/administração & dosagem , Proteínas de Bactérias/metabolismo , Infecções/tratamento farmacológico , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Via Secretória/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Bactérias/patogenicidade , Proteínas de Bactérias/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Infecções/patologia , Proteínas de Membrana/antagonistas & inibidores , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC , Proteínas SecARESUMO
Implant-related infections are a serious complication in prosthetic surgery, substantially jeopardizing implant fixation. As porous coatings for improved osseointegration typically present an increased surface roughness, their resulting large surface area (sometimes increasing with over 700% compared to an ideal plane) renders the implant extremely susceptible to bacterial colonization and subsequent biofilm formation. Therefore, there is particular interest in orthopaedic implantology to engineer surfaces that combine both the ability to improve osseointegration and at the same time reduce the infection risk. As part of this orthopaedic coating development, the interest of in vitro studies on the interaction between implant surfaces and bacteria/biofilms is growing. In this study, the in vitro staphylococcal adhesion and biofilm formation on newly developed porous pure Ti coatings with 50% porosity and pore sizes up to 50 µm is compared to various dense and porous Ti or Ti-6Al-4V reference surfaces. Multiple linear regression analysis indicates that surface roughness and hydrophobicity are the main determinants for bacterial adherence. Accordingly, the novel coatings display a significant reduction of up to five times less bacterial surface colonization when compared to a commercial state-of-the-art vacuum plasma sprayed coating. However, the results also show that a further expansion of the porosity with over 15% and/or the pore size up to 150 µm is correlated to a significant increase in the roughness parameters resulting in an ascent of bacterial attachment. Chemically modifying the Ti surface in order to improve its hydrophilicity, while preserving the average roughness, is found to strongly decrease bacteria quantities, indicating the importance of surface functionalization to reduce the infection risk of porous coatings.
Assuntos
Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Titânio/química , Porosidade , MolhabilidadeRESUMO
The metabolic impact exerted on a microorganism due to heterologous protein production is still poorly understood in Streptomyces lividans. In this present paper, based on exometabolomic data, a proposed genome-scale metabolic network model is used to assess this metabolic impact in S. lividans. Constraint-based modeling results obtained in this work revealed that the metabolic impact due to heterologous protein production is widely distributed in the genome of S. lividans, causing both slow substrate assimilation and a shift in active pathways. Exchange fluxes that are critical for model performance have been identified for metabolites of mouse tumor necrosis factor, histidine, valine and lysine, as well as biomass. Our results unravel the interaction of heterologous protein production with intracellular metabolism of S. lividans, thus, a possible basis for further studies in relieving the metabolic burden via metabolic or bioprocess engineering.
Assuntos
Genoma/fisiologia , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Streptomyces lividans/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , CamundongosRESUMO
The complete genome sequence of the original isolate of the model actinomycete Streptomyces lividans 66, also referred to as 1326, was deciphered after a combination of next-generation sequencing platforms and a hybrid assembly pipeline. Comparative analysis of the genomes of S. lividans 66 and closely related strains, including S. coelicolor M145 and S. lividans TK24, was used to identify strain-specific genes. The genetic diversity identified included a large genomic island with a mosaic structure, present in S. lividans 66 but not in the strain TK24. Sequence analyses showed that this genomic island has an anomalous (G + C) content, suggesting recent acquisition and that it is rich in metal-related genes. Sequences previously linked to a mobile conjugative element, termed plasmid SLP3 and defined here as a 94 kb region, could also be identified within this locus. Transcriptional analysis of the response of S. lividans 66 to copper was used to corroborate a role of this large genomic island, including two SLP3-borne "cryptic" peptide biosynthetic gene clusters, in metal homeostasis. Notably, one of these predicted biosynthetic systems includes an unprecedented nonribosomal peptide synthetase--tRNA-dependent transferase biosynthetic hybrid organization. This observation implies the recruitment of members of the leucyl/phenylalanyl-tRNA-protein transferase family to catalyze peptide bond formation within the biosynthesis of natural products. Thus, the genome sequence of S. lividans 66 not only explains long-standing genetic and phenotypic differences but also opens the door for further in-depth comparative genomic analyses of model Streptomyces strains, as well as for the discovery of novel natural products following genome-mining approaches.
Assuntos
Cobre/metabolismo , Ilhas Genômicas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA de Transferência/genética , Streptomyces lividans/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Peptídeos/metabolismo , Streptomyces coelicolor/genética , Streptomyces lividans/metabolismoRESUMO
Streptomyces lividans has shown potential as an expression system for heterologous proteins. Overexpression of proteic factors important for heterologous protein production is a valuable approach to improve yields of such proteins. Comparative transcriptomic analysis revealed that several genes were differentially expressed in strains involved in heterologous protein production. For instance, the gene-encoding phosphoenolpyruvate carboxykinase (pepck) showed a significant twofold change in recombinant S. lividans producing human tumour necrosis factor-alpha (hTNF-α). The effect of pepck overexpression on S. lividans TK24 and its hTNF-α producing recombinant was thus investigated in bench-top fermenters. Results obtained revealed that pepck overexpression resulted into a twofold increase in specific PEPCK activity during growth. This overexpression is correlated with slower growth rate, reduced excretion of pyruvate and less alkalinisation of the growth medium when compared with the control strain. After 26 h of fermentation, hTNF-α yields were enhanced (up to 1.7-fold) in the pepck-overexpressing S. lividans TK24, demonstrating that this metabolic engineering approach is indeed promising for heterologous protein production.
Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Streptomyces coelicolor/enzimologia , Streptomyces lividans/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/genética , Proteínas de Bactérias/metabolismo , Humanos , Engenharia Metabólica , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Streptomyces coelicolor/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective, thus causing damage to both healthy and cancerous tissue. Past research has focused on understanding tumour cells themselves, and conventional wisdom has aimed at targeting these cells directly. Recent research has shifted towards understanding the tumour microenvironment and it's differences from that of healthy cells/tissues in the body and then to exploit these differences for treatmeat of the tumour. One such approach is utilizing anaerobic bacteria. Several strains of bacteria have been shown to selectively colonize in solid tumours, making them valuable tools for selective tumour targeting and destruction. Amongst them, the anaerobic Clostridium has shown great potential in penetration and colonization of the hypoxic and necrotic areas of the tumour microenvironment, causing significant oncolysis as well as enabling the delivery of therapeutics directly to the tumour in situ. Various strategies utilizing Clostridium are currently being investigated, and represent a novel area of emerging cancer therapy. This review provides an update review of tumour microenvironment as well as summary of the progresses and current status of Clostridial spore-based cancer therapies.
RESUMO
Ribosome-inactivating proteins (RIPs) are toxic due to their N-glycosidase activity catalyzing depurination at the universally conserved α-sarcin loop of the 60S ribosomal subunit. In addition, RIPs have been shown to also have other enzymatic activities, including polynucleotide:adenosine glycosidase activity. RIPs are mainly produced by different plant species, but are additionally found in a number of bacteria, fungi, algae and some mammalian tissues. This review describes the occurrence of RIPs, with special emphasis on bacterial RIPs, including the Shiga toxin and RIP in Streptomyces coelicolor recently identified in S. coelicolor. The properties of RIPs, such as enzymatic activity and targeting specificity, and how their unique biological activity could be potentially turned into medical or agricultural tools to combat tumors, viruses and fungi, are highlighted.
Assuntos
Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/uso terapêutico , Proteínas Inativadoras de Ribossomos/toxicidade , Proteínas Inativadoras de Ribossomos/uso terapêutico , Proteínas de Algas/toxicidade , Proteínas Fúngicas/toxicidade , Humanos , Proteínas de Plantas/toxicidade , Shigella/metabolismo , Streptomyces coelicolor/metabolismoRESUMO
The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/química , Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Terciária de Proteína , Canais de Translocação SEC , Proteínas SecA , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ~27,000 compounds and proved to be highly reliable (average Z' factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Humanos , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC , Proteínas SecARESUMO
An efficient synthesis of a library of 5-amino-thiazolo[4,5-d]pyrimidines is reported. Regioselective displacements of chlorines, as well as regioselective diazotation reactions are described, which allow the introduction of structural diversity on the scaffold by consecutive reactions. Screening of this focused library led to the discovery of SecA inhibitors from Escherichia coli and Staphylococcus aureus.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Escherichia coli/enzimologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Staphylococcus aureus/enzimologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Proteínas de Membrana Transportadoras , Canais de Translocação SEC , Proteínas SecARESUMO
Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor. In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered. Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities. The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.
Assuntos
Aminoácidos/farmacocinética , Biotecnologia/métodos , Fermentação/fisiologia , Biossíntese de Proteínas/fisiologia , Streptomyces lividans/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Ácido Aspártico/metabolismo , Biomassa , Ácido Glutâmico/metabolismo , Metaboloma/genética , Camundongos , Especificidade da Espécie , Streptomyces lividans/fisiologiaRESUMO
Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of ß- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Streptomyces coelicolor/genética , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Glicosídeo Hidrolases/metabolismo , Biossíntese de Proteínas , Coelhos , Reticulócitos/metabolismo , Proteínas Inativadoras de Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMO
We compared the gas chromatography-mass spectrometry (GC-MS) metabolite profiles of mouse tumour necrosis factor alpha (mTNF-alpha) secreting Streptomyces lividans TK24 to the non-secreting wild type and the wild type harbouring the empty pIJ486 plasmid by multi-block principal component analysis (PCA). The multi-block PCA model successfully identified peaks that were statistically different between the protein secreting and non-secreting strains, and at the same time also uncovered the efficiency of intracellular metabolite extraction by an ultrasonic adaptive focused acoustics (AFA) technique compared to a manual vortex/freeze-thaw method. Fifty-one metabolites were significantly different between the three biological strains and 17 of these were abundant in the mTNF-alpha secreting strain compared to the non-secreting strains. No significant differences in the number of detected metabolite peaks were observed between the two extraction techniques. However, from the loadings of the multi-block PCA model, as well as univariate statistical analysis, we observed that the relative peak response ratios to the internal standard of 10 metabolites were higher for the AFA extraction, suggesting a more efficient recovery of these metabolites than achieved with the manual vortex/freeze thaw method.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Streptomyces lividans/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Aminoácidos/metabolismo , Animais , Metaboloma , Camundongos , Análise de Componente Principal , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces lividans/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Bacterial type I signal peptidase is a potential target for the development of novel antibacterial agents. In this study we demonstrate that a substrate based peptide aldehyde inhibits signal peptidases with a lower IC(50) value than the lipopeptides described to date. The length of the core lipopeptide could be reduced by removing several amino acids from both termini. Conversion of this peptide to an aldehyde resulted in a molecule with an IC(50) value of 0.09microM when tested against Staphylococcus [corrected] aureus SPase I, SpsB.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/metabolismo , Peptídeos/farmacologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Antibacterianos/síntese química , Antibacterianos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Peptídeos/síntese química , Peptídeos/química , Saccharomyces/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Twitching motility is a form of bacterial translocation over solid or semi-solid surfaces mediated by the extension, tethering, and subsequent retraction of type IV pili. These pili are also known to be involved in virulence, biofilm formation, formation of fruiting bodies, horizontal gene transfer, and protein secretion. We have characterized the presence of twitching motility on agar plates in Legionella pneumophila, the etiological agent of Legionnaires' disease. By examining twitching motility zones, we have demonstrated that twitching motility was dependent on agar thickness/concentration, the chemical composition of the media, the presence of charcoal and cysteine, proximity to other bacteria, and temperature. A knockout mutant of the pilus subunit, pilE, exhibited a total loss of twitching motility at 37 degrees C, but not at 27 degrees C, suggesting either the existence of a compensating pilus subunit or of another twitching motility system in this organism.