RESUMEN
The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas Biosensibles , Proteínas de Unión al ADN , Oxígeno/análisis , Pseudomonas fluorescens/genética , Microbiología del Suelo , Suelo/análisis , Transactivadores , Factores de Transcripción/genética , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Hordeum , Operón Lac , Oxígeno/metabolismo , Raíces de Plantas/microbiología , Regiones Promotoras Genéticas , Pseudomonas fluorescens/crecimiento & desarrollo , Pseudomonas fluorescens/metabolismoRESUMEN
Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Antiportadores/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Proteínas Represoras/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Adenosina Trifosfato/biosíntesis , Anaerobiosis , Antiportadores/biosíntesis , Arginina/farmacología , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Sitios de Unión , Regulación Bacteriana de la Expresión Génica , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.
Asunto(s)
4-Butirolactona/análogos & derivados , Proteínas Bacterianas/genética , Cianuros/metabolismo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , 4-Butirolactona/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Lipasa/biosíntesis , Datos de Secuencia Molecular , Plásmidos , Alineación de Secuencia , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Antiportadores/genética , Proteínas Bacterianas/genética , Lipasa/biosíntesis , Operón , Pseudomonas aeruginosa/genética , Proteínas Represoras , Anaerobiosis , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Proteínas de Escherichia coli , Genes Reguladores , Vectores Genéticos , Operón Lac , Datos de Secuencia MolecularRESUMEN
We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.