RESUMEN
To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.
Asunto(s)
Ácidos/farmacología , Adaptación Biológica/genética , Perfilación de la Expresión Génica/métodos , Helicobacter pylori/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Regulación hacia Abajo , Genoma Bacteriano , Concentración de Iones de Hidrógeno , Sistemas de Lectura Abierta , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Sensibilidad y Especificidad , Regulación hacia ArribaRESUMEN
The reporter bacterium, Pseudomonas fluorescens HK44 (HK44), was characterized in an immobilized state to investigate utility for deployment as a remote sensor in the subsurface. A packed-bed reactor with alginate-immobilized HK44 simulated hydrodynamic conditions such as might be found in a subsurface environment. The reporter bacterium, HK44, harbors a reporter plasmid, pUTK21, which contains a transcriptional fusion between the nahG gene in the lower pathway of the catabolic plasmic NAH7 and a luxCDABE gene cassette. The upper nah pathway and the lux pathway in pUTK21 are induced by salicylate. The lux enzymes catalyze the light reaction. HK44 demonstrated a quantitative relationship between salicylate concentration and degradation. Light intensity mimicked salicylate concentration, whereas degradation was first order in biomass and first order in salicylate concentration, with a degradation constant of 2.23 x 10(-2) dm(3) g(-1) min(-1).
RESUMEN
Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.