RESUMO
Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.
Assuntos
Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Variação Genética , Pichia/genética , Proteínas/genética , Retículo Endoplasmático/metabolismo , Dosagem de Genes , Humanos , Pichia/fisiologia , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica/genéticaRESUMO
A key component to the success of Mycobacterium tuberculosis as a pathogen is the ability to sense and adapt metabolically to the diverse range of conditions encountered in vivo, such as oxygen tension, environmental pH and nutrient availability. Although nitrogen is an essential nutrient for every organism, little is known about the genes and pathways responsible for nitrogen assimilation in M. tuberculosis. In this study we have used transcriptomics and chromatin immunoprecipitation and high-throughput sequencing to address this. In response to nitrogen starvation, a total of 185 genes were significantly differentially expressed (96 up-regulated and 89 down regulated; 5% genome) highlighting several significant areas of metabolic change during nitrogen limitation such as nitrate/nitrite metabolism, aspartate metabolism and changes in cell wall biosynthesis. We identify GlnR as a regulator involved in the nitrogen response, controlling the expression of at least 33 genes in response to nitrogen limitation. We identify a consensus GlnR binding site and relate its location to known transcriptional start sites. We also show that the GlnR response regulator plays a very different role in M. tuberculosis to that in non-pathogenic mycobacteria, controlling genes involved in nitric oxide detoxification and intracellular survival instead of genes involved in nitrogen scavenging.
Assuntos
Proteínas de Bactérias/metabolismo , Redes e Vias Metabólicas , Mycobacterium tuberculosis/metabolismo , Nitrogênio/metabolismo , Compostos de Amônio/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Parede Celular/metabolismo , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/genética , Ligação Proteica , Elementos de Resposta , Estresse FisiológicoRESUMO
BACKGROUND: Today's biological experiments often involve the collaboration of multidisciplinary researchers utilising several high throughput 'omics platforms. There is a requirement for the details of the experiment to be adequately described using standardised ontologies to enable data preservation, the analysis of the data and to facilitate the export of the data to public repositories. However there are a bewildering number of ontologies, controlled vocabularies, and minimum standards available for use to describe experiments. There is a need for user-friendly software tools to aid laboratory scientists in capturing the experimental information. RESULTS: A web application called XperimentR has been developed for use by laboratory scientists, consisting of a browser-based interface and server-side components which provide an intuitive platform for capturing and sharing experimental metadata. Information recorded includes details about the biological samples, procedures, protocols, and experimental technologies, all of which can be easily annotated using the appropriate ontologies. Files and raw data can be imported and associated with the biological samples via the interface, from either users' computers, or commonly used open-source data repositories. Experiments can be shared with other users, and experiments can be exported in the standard ISA-Tab format for deposition in public databases. XperimentR is freely available and can be installed natively or by using a provided pre-configured Virtual Machine. A guest system is also available for trial purposes. CONCLUSION: We present a web based software application to aid the laboratory scientist to capture, describe and share details about their experiments.
Assuntos
Projetos de Pesquisa , Software , Sistemas de Informação , Internet , Pessoal de Laboratório , Pesquisa , Pesquisadores , Interface Usuário-Computador , Vocabulário ControladoRESUMO
BACKGROUND: Nitrogen is an essential element for bacterial growth and an important component of biological macromolecules. Consequently, responding to nitrogen limitation is critical for bacterial survival and involves the interplay of signalling pathways and transcriptional regulation of nitrogen assimilation and scavenging genes. In the soil dwelling saprophyte Mycobacterium smegmatis the OmpR-type response regulator GlnR is thought to mediate the transcriptomic response to nitrogen limitation. However, to date only ten genes have been shown to be in the GlnR regulon, a vastly reduced number compared to other organisms. RESULTS: We investigated the role of GlnR in the nitrogen limitation response and determined the entire GlnR regulon, by combining expression profiling of M. smegmatis wild type and glnR deletion mutant, with GlnR-specific chromatin immunoprecipitation and high throughput sequencing. We identify 53 GlnR binding sites during nitrogen limitation that control the expression of over 100 genes, demonstrating that GlnR is the regulator controlling the assimilation and utilisation of nitrogen. We also determine a consensus GlnR binding motif and identify key residues within the motif that are required for specific GlnR binding. CONCLUSIONS: We have demonstrated that GlnR is the global nitrogen response regulator in M. smegmatis, directly regulating the expression of more than 100 genes. GlnR controls key nitrogen stress survival processes including primary nitrogen metabolism pathways, the ability to utilise nitrate and urea as alternative nitrogen sources, and the potential to use cellular components to provide a source of ammonium. These studies further our understanding of how mycobacteria survive nutrient limiting conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Genômica/métodos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Nitrogênio/metabolismo , Regulon/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Imunoprecipitação da Cromatina , Sequência Consenso/genética , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Motivos de Nucleotídeos/genética , TranscriptomaRESUMO
BACKGROUND: The ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown. RESULTS: A global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation. CONCLUSIONS: Application of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments.
Assuntos
Perfilação da Expressão Gênica , Genômica/métodos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/fisiologia , Nitrogênio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Algoritmos , Genoma Bacteriano/genética , Peróxido de Hidrogênio/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismoRESUMO
RESULTS: We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. CONCLUSION: Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.
Assuntos
Adaptação Fisiológica/genética , Regulação Fúngica da Expressão Gênica , Metanol/farmacologia , Pichia/genética , Biossíntese de Proteínas , Tripsina/genética , Técnicas de Cultura Celular por Lotes , Meios de Cultura , Variações do Número de Cópias de DNA , Humanos , Metanol/metabolismo , Oxirredução , Estresse Oxidativo , Pichia/efeitos dos fármacos , Pichia/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Engenharia de Proteínas , Transgenes , Tripsina/biossíntese , Resposta a Proteínas não DobradasRESUMO
PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Nitrogênio/deficiência , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Proteínas de Bactérias/fisiologia , Sistema Livre de Células/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos , Glutamato-Amônia Ligase/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/fisiologia , Processamento de Proteína Pós-Traducional , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Espectrometria de Massas em Tandem/métodosRESUMO
Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.