Asunto(s)
Analgésicos Opioides/efectos adversos , Agencias Gubernamentales , Pulmón/efectos de los fármacos , Contramedidas Médicas , Naloxona/uso terapéutico , Antagonistas de Narcóticos/uso terapéutico , Epidemia de Opioides , Trastornos Relacionados con Opioides/complicaciones , Insuficiencia Respiratoria/terapia , Conducta Cooperativa , Humanos , Comunicación Interdisciplinaria , Pulmón/fisiopatología , Naloxona/efectos adversos , Antagonistas de Narcóticos/efectos adversos , Epidemia de Opioides/mortalidad , Trastornos Relacionados con Opioides/mortalidad , Pronóstico , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/mortalidad , Insuficiencia Respiratoria/fisiopatología , Medición de Riesgo , Factores de Riesgo , Estados UnidosRESUMEN
The inherent architectural and chemical complexities of microbial biofilms mask our understanding of how these communities form, survive, propagate, and influence their surrounding environment. Here we describe a simple and versatile workflow for the cultivation and characterization of model flow-cell-based microbial ecosystems. A customized low-shear drip flow reactor was designed and employed to cultivate single and coculture flow-cell biofilms at the air-liquid interface of several metal surfaces. Pseudomonas putida F1 and Shewanella oneidensis MR-1 were selected as model organisms for this study. The utility and versatility of this platform was demonstrated via the application of several chemical and morphological imaging techniques-including matrix-assisted laser desorption/ionization mass spectrometry imaging, secondary ion mass spectrometry imaging, and scanning electron microscopy-and through the examination of model systems grown on iron substrates of varying compositions. Implementation of these techniques in combination with tandem mass spectrometry and a two-step imaging principal component analysis strategy resulted in the identification and characterization of 23 lipids and 3 oligosaccharides in P. putida F1 biofilms, the discovery of interaction-specific analytes, and the observation of several variations in cell and substrate morphology present during microbially influenced corrosion. The presented workflow is well-suited for examination of both single and multispecies drip flow biofilms and offers a platform for fundamental inquiries into biofilm formation, microbe-microbe interactions, and microbially influenced corrosion.
Asunto(s)
Biopelículas , Lípidos/análisis , Oligosacáridos/análisis , Imagen Óptica , Pseudomonas putida/metabolismo , Shewanella/metabolismo , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Oligosacáridos/metabolismo , Pseudomonas putida/química , Shewanella/químicaRESUMEN
Fatty acids are central to brain metabolism and signaling, but their distributions within complex brain circuits have been difficult to study. Here we applied an emerging technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS), to image specific fatty acids in a favorable model system for chemical analyses of brain circuits, the zebra finch (Taeniopygia guttata). The zebra finch, a songbird, produces complex learned vocalizations under the control of an interconnected set of discrete, dedicated brain nuclei 'song nuclei'. Using ToF-SIMS, the major song nuclei were visualized by virtue of differences in their content of essential and non-essential fatty acids. Essential fatty acids (arachidonic acid and docosahexaenoic acid) showed distinctive distributions across the song nuclei, and the 18-carbon fatty acids stearate and oleate discriminated the different core and shell subregions of the lateral magnocellular nucleus of the anterior nidopallium. Principal component analysis of the spectral data set provided further evidence of chemical distinctions between the song nuclei. By analyzing the robust nucleus of the arcopallium at three different ages during juvenile song learning, we obtain the first direct evidence of changes in lipid content that correlate with progression of song learning. The results demonstrate the value of ToF-SIMS to study lipids in a favorable model system for probing the function of lipids in brain organization, development and function.
Asunto(s)
Ácidos Grasos/metabolismo , Pinzones/fisiología , Vocalización Animal/fisiología , Animales , Química Encefálica/fisiología , Ácidos Grasos Esenciales/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Metabolismo de los Lípidos/fisiología , Masculino , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Análisis de Componente Principal , Prosencéfalo/metabolismo , Prosencéfalo/fisiología , Espectrometría de Masa de Ion SecundarioRESUMEN
Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the gamma-glutamyl transferase (gamma-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.
Asunto(s)
Análisis Mutacional de ADN , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Ruminococcus/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Subunidades de Proteína , ARN Bacteriano/análisis , ARN Mensajero/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ruminococcus/genética , Homología de Secuencia de AminoácidoRESUMEN
A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.
Asunto(s)
Bacteriófago T4/genética , Péptidos/genética , Empalme de Proteína , Selección Genética , Secuencia de Aminoácidos , Bacteriófago T4/enzimología , Bacteriófago T4/fisiología , Secuencia de Bases , Clonación Molecular , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Escherichia coli/virología , Datos de Secuencia Molecular , Péptidos/química , Proteínas Virales/genéticaRESUMEN
A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.