RESUMO
BACKGROUND: Streptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA. RESULTS: In this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21. CONCLUSIONS: Adaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.
Assuntos
Proteínas de Bactérias/metabolismo , Análise do Fluxo Metabólico/métodos , Streptomyces lividans/crescimento & desenvolvimento , Engenharia Metabólica , Modelos Teóricos , Transporte Proteico , Streptomyces lividans/metabolismoRESUMO
BACKGROUND: Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction. RESULTS: We have identified four acting thiol-disulphide oxidoreductases (TDORs) in S. lividans TK21, mutants in all TDOR candidates affect the secretion and activity of the Sec-dependent alpha-amylase, which contains several disulphide bonds, but the effect was more drastic in the case of the Sli-DsbA deficient strain. Thus, the four TDOR are required to obtain active alpha-amylase. Additionally, only mutations in Sli-DsbA and Sli-DsbB affect the secretion and activity of the Tat-dependent agarase, which does not form a disulphide bond, when it is overproduced. This suggests a possible role of the oxidised Sli-DsbA as a chaperone in the production of active agarase. CONCLUSIONS: Enzymes involved in the production of the extracellular mature active proteins are not fully characterised yet in Streptomyces lividans. Our results suggest that the role of thiol-disulphide oxidoreductases must be considered when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application, irrespective of their secretion route, in order to obtain active, correctly folded proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Dissulfetos/metabolismo , Oxirredutases/metabolismo , Streptomyces lividans/enzimologia , Regulação Bacteriana da Expressão Gênica , Domínios Proteicos , Dobramento de Proteína , Streptomyces lividans/genéticaRESUMO
BACKGROUND: Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. RESULTS: We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction. CONCLUSIONS: This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.
Assuntos
Glicosídeo Hidrolases/metabolismo , Streptomyces lividans/metabolismo , alfa-Amilases/metabolismo , Proteínas de Bactérias/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Modelos Biológicos , Dobramento de ProteínaRESUMO
Bacterial lipoproteins are a specialised class of membrane proteins that represent a small percentage of the proteome of Gram-positive bacteria, yet these lipoproteins have been reported to play important roles in nutrient scavenging, cell envelope assembly, protein folding, environmental signalling, host cell adhesion and virulence. Upon translocation of lipoproteins, the type II signal peptidase (Lsp) cleaves the signal peptide, leaving the lipoproteins bound to the outer face of the cytoplasmic membrane by means of linking lipid molecule to their +1 cysteine residue. We have studied the role played by Lsp in Streptomyces lividans cellular metabolism, particularly, in secretory protein production, and found that the absence of functional Lsp, apparently produces a translocase blockage, diminishes the synthesis of secretory proteins and triggers a stringent response. These findings could be particularly relevant when optimising S. lividans for the overproduction of secretory proteins of industrial application.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Lipoproteínas/metabolismo , Streptomyces lividans/enzimologia , Streptomyces lividans/genética , Transcrição Gênica , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Sinais Direcionadores de Proteínas , Transporte Proteico , Streptomyces lividans/metabolismoRESUMO
BACKGROUND: Next generation sequencing (NGS) enables a more comprehensive analysis of bacterial diversity from complex environmental samples. NGS data can be analysed using a variety of workflows. We test several simple and complex workflows, including frequently used as well as recently published tools, and report on their respective accuracy and efficiency under various conditions covering different sequence lengths, number of sequences and real world experimental data from rhizobacterial populations of glyphosate-tolerant maize treated or untreated with two different herbicides representative of differential diversity studies. RESULTS: Alignment and distance calculations affect OTU estimations, and multiple sequence alignment exerts a major impact on the computational time needed. Generally speaking, most of the analyses produced consistent results that may be used to assess differential diversity changes, however, dataset characteristics dictate which workflow should be preferred in each case. CONCLUSIONS: When estimating bacterial diversity, ESPRIT as well as the web-based workflow, RDP pyrosequencing pipeline, produced good results in all circumstances, however, its computational requirements can make method-combination workflows more attractive, depending on sequence variability, number and length.
Assuntos
Bactérias/classificação , Bactérias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiologia do Solo , Biodiversidade , DNA Ribossômico/genética , Humanos , RNA Ribossômico 16S/genética , Análise de Regressão , Análise de Sequência de DNA/métodos , Fluxo de TrabalhoRESUMO
A comparison was made of the effect of glyphosate (RoundupPlus), a post-emergency applied herbicide, and of HarnessGTZ, a pre-emergency applied herbicide, on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. The potential effect was monitored by direct amplification, cloning and sequencing of soil DNA encoding 16S rRNA, rhizobacterial DNA hybridization to commercially available genome-wide microarrays from the soil bacterium Streptomyces coelicolor, and high-throughput DNA pyrosequencing of the bacterial DNA coding for 16S rRNA hypervariable V6 region. The results obtained strongly suggest that both herbicides do in fact affect the maize rhizobacterial communities, glyphosate being, to a great extent, the environmentally less aggressive herbicide.
Assuntos
Glicina/análogos & derivados , Herbicidas/farmacologia , Microbiologia do Solo , Streptomyces coelicolor/efeitos dos fármacos , Zea mays/microbiologia , DNA Bacteriano/genética , Genoma Bacteriano , Glicina/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas/microbiologia , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Solo/análise , Poluentes do Solo/farmacologia , Streptomyces coelicolor/genética , Toluidinas/farmacologia , Triazinas/farmacologia , GlifosatoRESUMO
Commercially available DNA microarrays containing genome-wide spotted oligonucleotides encompass the soil bacteria Bacillus subtilis or Streptomyces coelicolor genomes. These have been used to analyse potential differences in rhizobacterial communities of transgenic maize engineered to express the Bacillus thuringensis Cry toxin (Bt maize) in three different agricultural soils. No differences in hybridisation were observed between genetically and non-genetically modified maize rhizobacteria from two Bt lines with a detection sensitivity of five copies of a particular gene above the background. Soil-specific hybridisation results were obtained when rhizobacterial DNA was compared to the corresponding genomic DNA spotted in the microarrays suggesting that the use of genome-wide DNA arrays could serve as a useful tool for the molecular monitoring of rhizobacterial communities.
Assuntos
Bacillus subtilis/genética , Microbiologia do Solo , Streptomyces coelicolor/genética , Zea mays/microbiologia , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Endotoxinas/genética , Genoma Bacteriano , Proteínas Hemolisinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Sensibilidade e Especificidade , Zea mays/genéticaRESUMO
Gram-positive soil bacteria included in the genus Streptomyces produce a large variety of secondary metabolites in addition to extracellular hydrolytic enzymes. From the industrial and commercial viewpoints, the S. lividans strain has generated greater interest as a host bacterium for the overproduction of homologous and heterologous hydrolytic enzymes as an industrial application, which has considerably increased scientific interest in the characterization of secretion routes in this bacterium. This review will focus on the secretion machinery in S. lividans.
RESUMO
Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application.
Assuntos
Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Streptomyces lividans/metabolismo , Peptídeo Hidrolases/genética , FosforilaçãoRESUMO
Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Glicosídeo Hidrolases/metabolismo , Streptomyces lividans/metabolismo , alfa-Amilases/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Glicosídeo Hidrolases/genética , Streptomyces lividans/genética , alfa-Amilases/genéticaRESUMO
Streptomyces lividans uses mainly two pathways to target secretory proteins to the cytoplasmic membrane. The major pathway (Sec pathway) transports pre-proteins using the signal recognition particle, and the minor Tat pathway is responsible for the secretion using a folded conformation of a relatively low number of proteins. The signal peptides of the Sec-dependent alpha-amylase and the Tat-dependent agarase were interchanged and fused in-frame to the corresponding mature part of the other enzyme. Alpha-amylase was unable to use the Tat route when fused to the agarase signal peptide, while agarase used the Sec route when it was targeted by the alpha-amylase signal peptide. In addition to the signal peptide some yet unidentified parts of the secreted proteins may play a role in selecting the secretory route. Structure predictions for the Tat- and Sec-dependent proteins suggest that less structured proteins are more likely to be candidates for the Tat route.
Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Streptomyces lividans/metabolismo , alfa-Amilases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Modelos Moleculares , Mutação , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Streptomyces lividans/genética , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
The Gram-positive eubacterium Streptomyces lividans contains four chromosomally encoded type I signal peptidases, SipW, SipX, SipY and SipZ, of which all but SipW have an unusual C-terminal membrane anchor. For in vitro characterisation of these signal peptidases, the S. lividans sip genes were expressed in Escherichia coli and the corresponding proteins were purified. The four enzymes had an optimum activity at an alkaline pH, notably pH 8-9 for SipW and SipY and pH 10-11 for SipX and SipZ. In contrast to SipW, the in vitro activities of SipX, SipY and SipZ significantly increased in the presence of detergent. Since none of the S. lividans Sip proteins contains the hydrophobic beta-barrel domain, which in E. coli LepB was proven to be requisite for detergent-dependent in vitro activity, we assume that for detergent dependence, the C-terminal transmembrane anchor can partly substitute for this domain. Finally, all Sip proteins were stimulated by added phospholipids, which strongly suggests that phospholipids play an important role in the catalytic mechanism.
Assuntos
Proteínas de Membrana , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Reports of herbicide resistance events are proliferating worldwide, leading to new cultivation strategies using combinations of pre-emergence and post-emergence herbicides. We analyzed the impact during a one-year cultivation cycle of several herbicide combinations on the rhizobacterial community of glyphosate-tolerant Bt-maize and compared them to those of the untreated or glyphosate-treated soils. Samples were analyzed using pyrosequencing of the V6 hypervariable region of the 16S rRNA gene. The sequences obtained were subjected to taxonomic, taxonomy-independent, and phylogeny-based diversity studies, followed by a statistical analysis using principal components analysis and hierarchical clustering with jackknife statistical validation. The resilience of the microbial communities was analyzed by comparing their relative composition at the end of the cultivation cycle. The bacterial communites from soil subjected to a combined treatment with mesotrione plus s-metolachlor followed by glyphosate were not statistically different from those treated with glyphosate or the untreated ones. The use of acetochlor plus terbuthylazine followed by glyphosate, and the use of aclonifen plus isoxaflutole followed by mesotrione clearly affected the resilience of their corresponding bacterial communities. The treatment with pethoxamid followed by glyphosate resulted in an intermediate effect. The use of glyphosate alone seems to be the less aggressive one for bacterial communities. Should a combined treatment be needed, the combination of mesotrione and s-metolachlor shows the next best final resilience. Our results show the relevance of comparative rhizobacterial community studies when novel combined herbicide treatments are deemed necessary to control weed growth..
Assuntos
Bactérias/efeitos dos fármacos , Herbicidas/farmacologia , Plantas Geneticamente Modificadas/microbiologia , Zea mays/microbiologia , Bactérias/classificação , Bactérias/genética , Análise por Conglomerados , DNA Bacteriano/genética , Metagenoma , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genéticaRESUMO
In this paper we have addressed the problem of analysing Next Generation Sequencing samples with an expected large biodiversity content. We analysed several well-known 16S rRNA datasets from experimental samples, including both large and short sequences, in numbers of tens of thousands, in addition to carefully crafted synthetic datasets containing more than 7000 OTUs. From this data analysis several patterns were identified and used to develop new guidelines for experimentation in conditions of high biodiversity. We analysed the suitability of different clustering packages for these type of situations, the problem of even sampling, the relative effectiveness of Chao1 and ACE estimators as well as their effect on sampling size for a variety of population distributions. As regards practical analysis procedures, we advocated an approach that retains as much high-quality experimental data as possible. By carefully applying selection rules combining the taxonomic assignment with clustering strategies, we derived a set of recommendations for ultra-sequencing data analysis at high biodiversity levels.
Assuntos
Biodiversidade , Metagenômica , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Sequenciamento de Nucleotídeos em Larga Escala , RNA Ribossômico 16S/genéticaRESUMO
The rhizobacterial composition varies according to the soil properties. To test if the effect of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations, a comparison was made between the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, and a pre-emergence applied herbicide (GTZ) versus untreated soil. The potential effect was monitored by direct amplification, cloning, and sequencing of the soil DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. The results obtained using three different methods to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 maize were comparable to those previously obtained when glyphosate-tolerant maize was grown in soil with different characteristics. Both herbicides decreased the bacterial diversity in the rhizosphere, with Actinobacteria being the taxonomic group most affected. The results suggest that both herbicides affected the structure of the maize rhizobacterial community, but glyphosate was environmentally less aggressive.
Assuntos
Bactérias/classificação , Bactérias/efeitos dos fármacos , Biota , Glicina/análogos & derivados , Herbicidas/metabolismo , Microbiologia do Solo , Zea mays/microbiologia , Bactérias/metabolismo , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Glicina/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Rizosfera , Análise de Sequência de DNA , GlifosatoRESUMO
BACKGROUND: Bacterial two-component signal transduction regulatory systems are the major set of signalling proteins frequently mediating responses to changes in the environment. They typically consist of a sensor, a membrane-associated histidine kinase and a cytoplasmic response regulator. The membrane-associated sensor detects the environmental signal or stress, whereas the cytoplasmic regulatory protein controls the cellular response usually by gene transcription modulation. METHODOLOGY/PRINCIPALFINDINGS: The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system, where SCO5784 encodes a histidine-kinase sensor and SCO5785 encodes a response regulator protein. When the expression level of the regulator gene decreases, the antibiotic synthesis and sporulation is delayed temporarily in addition to some ribosomal genes became up regulated, whereas the propagation of the regulatory gene in high copy number results in the earlier synthesis of antibiotics and sporulation, as well as the down regulation of some ribosomal genes and, moreover, in the overproduction of several extracellular proteins. Therefore, this two-component system in S. coelicolor seems to influence various processes characterised by the transition from primary to secondary metabolism, as determined by proteomic and transcriptomic analyses. CONCLUSIONS/SIGNIFICANCE: Propagation of SCO5785 in multicopy enhances the production of antibiotics as well as secretory proteins. In particular, the increase in the expression level of secretory protein encoding genes, either as an artefactual or real effect of the regulator, could be of potential usefulness when using Streptomyces strains as hosts for homologous or heterologous extracellular protein production.
Assuntos
Transdução de Sinais/fisiologia , Streptomyces coelicolor/metabolismo , Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Metabolismo , ÓperonRESUMO
BACKGROUND: Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins. METHODOLOGY/PRINCIPAL FINDINGS: Overproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response. CONCLUSIONS/SIGNIFICANCE: To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.
Assuntos
Streptomyces lividans/metabolismo , Estresse Fisiológico/fisiologia , alfa-Amilases/metabolismo , Regulação Bacteriana da Expressão Gênica , Dobramento de Proteína , Streptomyces lividans/genética , alfa-Amilases/genéticaRESUMO
BACKGROUND: Bt-maize is a transgenic variety of maize expressing the Cry toxin from Bacillus turingiensis. The potential accumulation of the relative effect of the transgenic modification and the cry toxin on the rhizobacterial communities of Bt-maize has been monitored over a period of four years. METHODOLOGY/PRINCIPAL FINDINGS: The accumulative effects of the cultivation of this transgenic plant have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The obtained sequences were subjected to taxonomic, phylogenetic and taxonomic-independent diversity studies. The results obtained were consistent, indicating that variations detected in the rhizobacterial community structure were possibly due to climatic factors rather than to the presence of the Bt-gene. No variations were observed in the diversity estimates between non-Bt and Bt-maize. CONCLUSIONS/SIGNIFICANCE: The cultivation of Bt-maize during the four-year period did not change the maize rhizobacterial communities when compared to those of the non-Bt maize. This is the first study to be conducted with Bt-maize during such a long cultivation period and the first evaluation of rhizobacterial communities to be performed in this transgenic plant using Next Generation Sequencing.
Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Microbiologia do Solo , Zea mays/crescimento & desenvolvimento , Toxinas de Bacillus thuringiensis , Bactérias/metabolismo , Proteínas de Bactérias/genética , Análise por Conglomerados , Endotoxinas/genética , Proteínas Hemolisinas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Análise de Sequência de DNA/métodos , Zea mays/genética , Zea mays/microbiologiaRESUMO
Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Serina Endopeptidases/metabolismo , Streptomyces lividans/enzimologia , Streptomyces lividans/crescimento & desenvolvimento , Proteínas de Membrana/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Serina Endopeptidases/genética , Streptomyces lividans/citologiaRESUMO
A comparison was drawn between the effect of glyphosate (Roundup Plus), a post-emergence applied herbicide, and Harness GTZ, a pre-emergence applied herbicide, on the culturable fraction of the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. Two different non-selective rich media were used to grow fast-growing culturable bacteria, BHI and NB, as a more accurate estimation of the soil fast-growing culturable bacterial population would be obtained from the results of cultivating in more than one medium. The potential effect was monitored by direct amplification, cloning and sequencing of bacterial DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from bacterial communities grown in the two different media. The estimated relative composition of the culturable maize rhizobacterial population varied considerably in accordance with the growth medium used. Both herbicides do, in fact, affect the maize rhizobacterial communities, glyphosate being, to a great extent, the less aggressive herbicide, regardless of the cultivation medium used. The pyrosequencing analysis of the fast-growing bacterial populations from the different soils represents a useful and invaluable tool to estimate the bacterial biodiversity of the culturable rhizobacteria of agricultural soils.