RESUMEN
Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B ⢠Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control ⢠Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.
Asunto(s)
Corynebacterium glutamicum , Aminoácidos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , AlemaniaRESUMEN
The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate-2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5' end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis.IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/fisiología , Deficiencias de Hierro , ARN Mensajero/metabolismo , Tiamina/biosíntesis , Corynebacterium glutamicum/crecimiento & desarrollo , Proteoma , TranscriptomaRESUMEN
BACKGROUND: Different polycystic ovary syndrome (PCOS) phenotypes are correlated with different clinical severity levels. Insulin resistance correlates with higher severity. In a retrospective study, 130 patients with polycystic ovary syndrome were examined for insulin resistance. The aim of the study was to investigate relationships between glucose metabolism and different PCOS phenotypes and to identify biomarkers or combinations thereof to obtain information on the type of metabolic disorder or the severity of PCOS. METHODS: A total of 130 patients with PCOS were included in the study. Biometric data such as weight, height, cycle day and cycle length were compared with glucose metabolism parameters such as fasting glucose, insulin before and 60 and 120 minutes after 75 g glucose intake, intact proinsulin, C-peptide and ovarian function parameters including Anti-Müllerian hormone (AMH) and the soluble AMH receptor (sAMHR2). The parameters were correlated, and their diagnostic performance with respect to different expressions of PCOS was evaluated. RESULTS: The biomarkers of impaired glucose metabolism showed strong significant difference in HOMA Index, adiponectin, proinsulin and body mass index (BMI) and Insulin levels in 0-60-120 minutes of glucose tolerance test but also with parameters of ovarian function as AMH, AMH z-score sAMHR2, and sAMHR2/AMH ratio. A strong correlation between sAMHR2 and adiponectin (r = .818, P < .0001) was found indicating a relationship between the degree of glucose metabolic impairment and ovarian function. CONCLUSIONS: The parameters glucose, insulin, insulin 60 minutes after intake of 75 g glucose and adiponectin or sAMHR2 enable a biochemical classification of PCOS patients that correlates with morphological PCOS phenotypes. By determining biomarkers, it is possible to classify PCOS patients into subgroups that correlate with different PCOS phenotypes and the clinical severity.
Asunto(s)
Resistencia a la Insulina , Síndrome del Ovario Poliquístico , Hormona Antimülleriana , Péptido C , Femenino , Glucosa , Humanos , Fenotipo , Estudios RetrospectivosRESUMEN
Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with d-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.
Asunto(s)
Biotecnología/métodos , Corynebacterium glutamicum/genética , Genoma Bacteriano , Biología Sintética/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Corynebacterium glutamicum/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Frecuencia de los Genes , Familia de Multigenes/genética , Fenotipo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Corynebacterium/metabolismo , Ferritinas/metabolismo , Homeostasis/fisiología , Hierro/metabolismo , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas Mutantes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteómica/métodos , Ubiquitinas/metabolismo , Proteínas Bacterianas/genética , Cromatografía Liquida , Corynebacterium glutamicum/genética , Bases de Datos de Proteínas , Mutación/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) CBS7435 is the parental strain of commonly used P. pastoris recombinant protein production hosts making it well suited for improving the understanding of associated genomic features. Here, we present a 9.35 Mbp high-quality genome sequence of P. pastoris CBS7435 established by a combination of 454 and Illumina sequencing. An automatic annotation of the genome sequence yielded 5007 protein-coding genes, 124 tRNAs and 29 rRNAs. Moreover, we report the complete DNA sequence of the first mitochondrial genome of a methylotrophic yeast. Fifteen genes encoding proteins, 2 rRNA and 25 tRNA loci were identified on the 35.7 kbp circular, mitochondrial DNA. Furthermore, the architecture of the putative alpha mating factor protein of P. pastoris CBS7435 turned out to be more complex than the corresponding protein of Saccharomyces cerevisiae.