RESUMO
RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.
Assuntos
Núcleo Celular/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Homólogo 5 da Proteína Cromobox/metabolismo , Cromossomos/metabolismo , DNA/metabolismo , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dactinomicina/farmacologia , Feminino , Genoma , Células HEK293 , Heterocromatina/metabolismo , Humanos , Camundongos , Modelos Biológicos , Família Multigênica , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA-DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video ( https://youtu.be/6SdWkBxQGlg ). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.
Assuntos
Núcleo Celular , Código de Barras de DNA Taxonômico/métodos , DNA , Genômica/métodos , Software , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/fisiologia , DNA/genética , DNA/metabolismo , Feminino , Técnicas Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , CamundongosRESUMO
Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study, we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present blue native-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in the knowledge of this organism's biology. On a more limited scale, we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecalis, which is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.
Assuntos
Proteínas de Bactérias/análise , Membrana Celular/química , Enterococcus faecalis/química , Proteoma/análise , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/química , Proteínas de Membrana/análise , Chaperonas Moleculares/análise , Complexos Multiproteicos/química , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel Bidimensional/métodos , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas/métodos , Proteínas de Membrana/classificação , Proteínas de Membrana/isolamento & purificação , Chaperonas Moleculares/classificação , Chaperonas Moleculares/isolamento & purificação , Peso Molecular , Complexos Multiproteicos/isolamento & purificação , Filogenia , Proteoma/análise , Proteômica/métodosRESUMO
Mitotic crossovers have the potential to cause large-scale genome rearrangements. Here, we describe high-throughput, single-cell, whole-genome sequencing methods for mapping crossovers genome-wide at scale. The methods are generalizable to various eukaryotes and to other end points requiring high-throughput, high-coverage single cell sequencing.
Assuntos
GenomaRESUMO
The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Pluripotentes/metabolismo , Cromatina/metabolismo , Metilação de DNA , Elementos Facilitadores Genéticos , Humanos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.
Assuntos
Anticorpos/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Animais , Formação de Anticorpos/imunologia , Fenômenos Biofísicos , Humanos , Camundongos KnockoutRESUMO
The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.
Assuntos
Arseniato Redutases/metabolismo , Ectothiorhodospiraceae/enzimologia , Oxirredutases/metabolismo , Sequência de Aminoácidos , Arseniato Redutases/classificação , Arseniato Redutases/genética , Ectothiorhodospiraceae/genética , Genoma Bacteriano , Dados de Sequência Molecular , Óperon , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , Shewanella/enzimologia , Shewanella/genéticaRESUMO
For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies use RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per-cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here, we describe VDJ sequencing (VDJ-seq), which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer-extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMIs) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and nonproductive VDJ and DJ recombination events on a per-cell basis. Library preparation takes 3 d, with 2 d of sequencing and 1 d of data processing and analysis.
Assuntos
Genes de Imunoglobulinas , Variação Genética , Imunoglobulinas/genética , Análise de Sequência de DNA/métodos , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , CamundongosRESUMO
BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.
Assuntos
Envelhecimento/genética , Linfócitos B/metabolismo , Cromatina/química , Epigênese Genética , Somatomedinas/fisiologia , Transcriptoma , Envelhecimento/imunologia , Animais , Linfócitos B/imunologia , Regulação para Baixo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.
RESUMO
Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.
Assuntos
Antígenos de Diferenciação/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/biossíntese , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.
Assuntos
Cromatina/metabolismo , Recombinação V(D)J/genética , Algoritmos , Animais , Epigênese Genética , Evolução Molecular , Regulação da Expressão Gênica , Loci Gênicos , Proteínas de Homeodomínio/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Receptores de Antígenos , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Alkaliphilus oremlandii strain OhILAs, a gram-positive bacterium, has been shown to ferment lactate as well as use arsenate and roxarsone as a terminal electron acceptor. This study examines the proteome expressed under four growth conditions to further elucidate the bacterial metabolism of inorganic and organic arsenic. The four growth conditions include, sodium lactate (as fermentative control), sodium lactate with 3-nitro-4-hydroxybenzenearsonic acid (roxarsone), sodium lactate with 3-amino-4-hydroxybenzenearsonic acid (3A4HBAA), and sodium lactate with sodium arsenate. Shotgun proteomics using LC-MS/MS was performed on the soluble cytoplasm as well as solubilized membrane proteins using perfluorooctanoic acid, a surfactant with properties similar to sodium dodecyl sulfate. The MS/MS data were analyzed using the Spectrum Mills Proteomic Workbench. Positive protein matches were confirmed with protein scores of 20 or greater and the presence of two or more peptides among the three technical replicates. A total of 1357 proteins (out of 2836 predicted) were identified with 791 in sodium lactate, 816 in sodium lactate and roxarsone, 715 in sodium lactate and 3A4HBAA, and 733 in sodium lactate and arsenate. The relative abundance of each protein was determined using a method called normalized spectral abundance factor (NSAF). Proteins that were identified in both the control and the experimental conditions were compared using the Power Law Global Error Model (PLGEM) to determine proteins that were significantly up or down regulated. All putative proteins were assigned functions and pathways using the COG databases. However, a large number of proteins were classified as hypothetical or had unknown function. Using the statistical information and known functionalities of the identified proteins, a pathway for the degradation of roxarsone and 3A4HBAA by A. oremlandii strain OhILAs is proposed.
Assuntos
Arsenicais/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium/metabolismo , Ácido Arsanílico/metabolismo , Arseniatos/metabolismo , Biotransformação , Clostridium/crescimento & desenvolvimento , Proteômica , Roxarsona/metabolismo , Espectrometria de Massas em TandemRESUMO
A major challenge for the bioremediation of toxic metals is the co-occurrence of nitrate, as it can inhibit metal transformation. Geobacter metallireducens, Desulfovibrio desulfuricans, and Sulfurospirillum barnesii are three soil bacteria that can reduce chromate [Cr(VI)] and nitrate, and may be beneficial for developing bioremediation strategies. All three organisms respire through dissimilatory nitrate reduction to ammonia (DNRA), employing different nitrate reductases but similar nitrite reductase (Nrf). G. metallireducens reduces nitrate to nitrite via the membrane bound nitrate reductase (Nar), while S. barnesii and D. desulfuricans strain 27774 have slightly different forms of periplasmic nitrate reductase (Nap). We investigated the effect of DNRA growth in the presence of Cr(VI) in these three organisms and the ability of each to reduce Cr(VI) to Cr(III), and found that each organisms responded differently. Growth of G. metallireducens on nitrate was completely inhibited by Cr(VI). Cultures of D. desulfuricans on nitrate media was initially delayed (48 h) in the presence of Cr(VI), but ultimately reached comparable cell yields to the non-treated control. This prolonged lag phase accompanied the transformation of Cr(VI) to Cr(III). Viable G. metallireducens cells could reduce Cr(VI), whereas Cr(VI) reduction by D. desulfuricans during growth, was mediated by a filterable and heat stable extracellular metabolite. S. barnesii growth on nitrate was not affected by Cr(VI), and Cr(VI) was reduced to Cr(III). However, Cr(VI) reduction activity in S. barnesii, was detected in both the cell free spent medium and cells, indicating both extracellular and cell associated mechanisms. Taken together, these results have demonstrated that Cr(VI) affects DNRA in the three organisms differently, and that each have a unique mechanism for Cr(VI) reduction.
RESUMO
Clostridial species predominate in both chicken gastrointestinal tract as well as litter where the organoarsenical roxarsone (3-nitro 4-hydroxybenzenearsonic acid) is anaerobically transformed releasing the more recognized toxic inorganic arsenic. 2D-gel electrophoresis and mass spectrometry were used to evaluate the changes in protein expression of Alkaliphilus oremlandii in response to different growth conditions (e.g., terminal electron acceptors) in order to explore the mechanism of microbial biotransformation of roxarsone. Aldehyde ferredoxin oxidoreductase, the enzyme that belongs to the xanthine oxidase family of molybdoenzymes was significantly overexpressed in the presence of roxarsone suggesting a role in the anaerobic metabolism of this substituted nitrophenol.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridium/metabolismo , Proteoma/metabolismo , Roxarsona/metabolismo , Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Biotransformação , Clostridium/enzimologia , Meios de Cultura , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Proteoma/química , Proteoma/efeitos dos fármacos , Roxarsona/farmacocinética , Roxarsona/farmacologia , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Identical masses of submerged Trichoderma viride mycelia of various ages were used as inoculum for a second submerged cultivation lasting for 24 h. It was found that the growth yield of secondary culture was dependent on the age of inoculum. The growth yields increased when the age of primary culture was less than 3 d, and decreased down to zero when older mycelia were inoculated. The mycelia were living even after 1 month of submerged cultivation, as they formed conidia after inoculating onto solid medium. In order to elucidate underlying biochemical processes, developmental changes of specific activities of organellar marker enzymes were measured in the mitochondrial/vacuolar and microsomal fractions of mycelia. These activities changed during the growth of mycelia in a biphasic manner and their time courses were remarkably similar. Only the H(+)-ATPase activity decreased monophasically with the age of mycelia. Membrane-bound proteases of both membrane fractions changed differently upon ageing. These results could not be explained as a consequence of nutrient starvation and indicate that the prolonged submerged cultivation triggers coordinated series of biochemical events which leads to the loss of growth competence.
Assuntos
Micélio/crescimento & desenvolvimento , Trichoderma/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Micélio/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Fatores de Tempo , Trichoderma/enzimologiaRESUMO
Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.
Assuntos
Proteínas de Ciclo Celular/farmacologia , Membrana Celular/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Oxirredutases/farmacologia , Western Blotting , Proteínas de Ciclo Celular/química , Divisão Celular , Grupo dos Citocromos b , Citocromos/metabolismo , Dimerização , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Íons/química , Substâncias Macromoleculares/química , Espectrometria de Massas , Proteínas de Membrana/química , Mutação , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Trichoderma viride was capable of growth and conidiation in the presence of high concentrations of the uncoupler 3,3',4',5-tetrachlorosalicylanilide (up to 100 micromol x L(-1) and of the respiration inhibitor mucidin (up to 100 micromol x L(-1) ) in both submerged and surface cultivation. When vegetative mycelia were cultivated on the solid Czapek-Dox medium with yeast autolysate under an anaerobic and CO2-containing atmosphere, the growth was observed only rarely but the microorganism survived as long as 3 months under these conditions. Major products of metabolism of both aerobic and anaerobic submerged mycelia were identified by means of 1H-NMR measurements. Major products excreted to the medium under aerobic conditions were succinic and citric acids. Major metabolites present in the submerged mycelia were gamma-aminobutyric (and glutamic) acid and alanine. Under anaerobic conditions, citric acid was not excreted into the medium but ethanol appeared. Its production could not be increased upon increasing the sugar concentration. The appearance of secondary metabolites was found to be modified by oxygen availability during the mycelial growth. Results suggest that the vegetative form of T. viride is capable of fermentative metabolism characterized by the production of ethanol and succinate and that the excretion of carboxylic acids is developmentally regulated and modified by oxygen availability.