Global responses of Methanococcus maripaludis to specific nutrient limitations and growth rate.

Continuous culture, transcriptome arrays, and measurements of cellular amino acid pools and tRNA charging levels were used to determine the response of Methanococcus maripaludis to leucine limitation. For comparison, the responses to phosphate and H2 limitations were measured as well. In addition, the effect of growth rate was determined. Leucine limitation resulted in a broad response. tRNA(Leu) charging decreased, but only small increases in mRNA were seen for amino acid biosynthesis genes. However, the cellular levels of free isoleucine and valine showed significant increases, indicating a coordinate regulation of branched-chain amino acids at a post-mRNA level. Leucine limitation also resulted in increased mRNA abundance for ribosomal protein genes, increased rRNA abundance, and decreased mRNA abundance for genes of methanogenesis. In contrast, phosphate limitation induced a specific response, a marked increase in mRNA levels for a phosphate transporter. Some mRNA levels responded to more than one factor; for example, transcripts for flagellum synthesis genes decreased under conditions of leucine limitation and increased under H2 limitation. Increased growth rate resulted in increased mRNA levels for ribosomal protein genes, increased rRNA abundance, and increased mRNA for a gene encoding an S-layer protein.

Biosynthesis of phosphoserine in the Methanococcales.

Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA(Cys) with l-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.

Orthologs of a novel archaeal and of the bacterial peptidyl-tRNA hydrolase are nonessential in yeast.

Peptidyl-tRNA hydrolase (encoded by pth) is an essential enzyme in all bacteria, where it releases tRNA from the premature translation termination product peptidyl-tRNA. Archaeal genomes lack a recognizable peptidyl-tRNA hydrolase (Pth) ortholog, although it is present in most eukaryotes. However, we detected Pth-like activity in extracts of the archaeon Methanocaldococcus jannaschii. The uncharacterized MJ0051 ORF was shown to correspond to a protein with Pth activity. Heterologously expressed MJ0051 enzyme catalyzed in vitro the cleavage of the Pth substrates diacetyl-[14C]lysyl-tRNA and acetyl-[14C]phenylalanyl-tRNA. On transformation of an Escherichia coli pth(ts) mutant, the MJ0051 gene (named pth2) rescued the temperature-sensitive phenotype of the strain. Analysis of known genomes revealed the presence of highly conserved orthologs of the archaeal pth2 gene in all archaea and eukaryotes but not in bacteria. The phylogeny of pth2 homologs suggests that the gene has been vertically inherited throughout the archaeal and eukaryal domains. Deletions in Saccharomyces cerevisiae of the pth2 (YBL057c) or pth (YHR189w) orthologs were viable, as was the double deletion strain, implying that the canonical Pth and Pth2 enzymes are not essential for yeast viability.

Búsqueda de Mycoplasma pneumoniae, M. hominis y M. fermentans en infecciones respiratorias / Searching for Mycoplasma pneumonia, Mycoplasma hominis, and Mycoplasma fermentans in respiratory infections

Mycoplasma pneumoniae es un microorganismo que produce un amplio espectro de síntomas respiratorios y otros síndromes clínicos. La neumonía micoplásmica es extremadamente variada, su inicio es de dos semanas después de la exposición y generalmente es gradual, con dolor de cabeza, fiebre, escalofrío, eritema de las membranas timpánicas y de la mucosa de la faringe posterior; en la mayoría de los casos se acompaña de tos no productiva. La imagern radiológica de la neumonía causada por esta bacteria no es característica, por lo que no se puede diferenciar de la originada por virus. La metodología utilizada en este trabajo se estandarizó con cepas de referencia, utilizando pruebas bioquímicas, fisiológicas y serológicas. Se estudiaron 157 muestras respiratorias de 145 enfermos. La neumonía y/o bronconeumonía fueron los diagnósticos clínicos en donde se encontró a Mycoplasma sp. con mayor frecuencia (46.2 por ciento), seguidas por neumonía complicada (20 por ciento). En total se obtuvieron 40 cepas de Mycoplasma sp.; de éstas correspondió a M. pneumoniae 13.1 por ciento, M. Hominis 9.6 por ciento y M. fermentans 4.8 por ciento. Se analizaron 60 sueros por la prueba de inhibición metabólica para determinar anticuerpos contra Mycoplasma sp.; ello permitió detectar a pacientes con títulos significativos de anticuerpos mayores de 1:32 que tuvieron cultivo negativo; sin esta prueba no se hubiera sospechado la presencia de la bacteria en el paciente; esto demuestra que, para obtener un diagnóstico más preciso, se requiere por lo menos del cultivo y la serología

Infecciones respiratorias: agentes etiológicos / Respiratory infections: etiologic agents

Se estudiaron 119 pacientes de los Departamentos de Neumología Pediátrica y de Adultos del Hospital General del Centro Médico Nacional La Raza (IMSS), con diagnóstico clínico y radiológico de neumonía/bronconeumonía (63 casos), bronquitis/enfisema pulmonar (31 casos) y neumonía complicada (25 casos). Se obtuvieron 125 muestras de secreciones broncopulmonares mediante técnicas invasivas y no-invasivas: aspirado bronquial (44), lavado broncoalveolar (20), cepillado bronquial (29), punción pulmonar (18) y expectoración (14). A todas las muestras se les practicaron estudios bacteriológicos, micológicos y a los anaerobios facultativos sensibilidad a los antimicrobianos. Además, se realizaron 100 hemocultivos. Se aislaron 211 cepas bacterianas. Gram negativas 37.2 por ciento, Gram positivas 26.1 por ciento, anaerobios obligados 13.7 por ciento, mycoplasma sp 13.3 por ciento y levaduras 10.9 por ciento. Entre las bacterias Gram negativas predominó Pseudomonas aeruginosa seguida por ocho géneros diferentes de enterobacterias. Prevotella melaninogenicus entre los anaerobios obligados. La levadura más frecuentemente aislada fue Candida albicans. Pseudomonas sp presentó elevada resistencia a los antimicrobianos y solamente un hemocultivo fue positivo a Staphylococcus epidermidis. Los agentes etiológicos aislados en este estudio, en general, fueron muy diversos además de presentarse en cultivo mixto.