RESUMEN
N-methyl-N'-nitro-N-nitrosoguanidine (NH)-induced mutagenesis with subsequent selection for resistance to toxic amino acid analogues (azaserine, m-fluoro-DL-phenylalanine, and 6-diazo-5-oxo-L-norleucine) was applied to Pseudomonas aurantiaca B-162. The resulting strains produced phenazine antibiotics three times more efficiently than the wild type strain and ten times more efficiently than the known pseudomonad strains. Overproduction of phenazine antibiotics was shown to result either from deregulation of 3-deoxi-D-arabinohepulosonate-7-phosphate synthase (DAHP synthase), the key enzyme of the aromatic pathway (removal of inhibition by phenylalanine, tyrosine, and phenazine), or overproduction of N-hexanoyl homoserine lactone, the regulatory molecule of positive control of cellular metabolism (QS system).
Asunto(s)
Fenazinas/metabolismo , Pseudomonas/aislamiento & purificación , Pseudomonas/metabolismo , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Azaserina/farmacología , Diazooxonorleucina/farmacología , Farmacorresistencia Bacteriana , Metilnitronitrosoguanidina , Mutagénesis , Fenilalanina/farmacología , Pseudomonas/efectos de los fármacos , Pseudomonas/genéticaRESUMEN
The regulation of activity and synthesis of DAHP-synthase in Pseudomonas aurantiaca B-162 was studied. Analysis of partially purified preparations of the enzyme revealed two isoenzymes: DAHP-synthase [tyr] and DAHP-synthase [phe], each of them being regulated by a corresponding amino acid. DAHP-synthase [tyr] is a dominant isoenzyme presenting 78 % of the enzyme activity, 50 % inhibition of which is possible by 1,3 x 10(-5) M of tyrosine. DAHP-synthase [phe] is minor isoenzyme (sharing 22 % of enzyme activity) and is controlled by phenylalanine. In this case, 50% of inhibition of activity is possible by adding 5,5 10(-6) M of corresponding amino acid. Synthesis of DAHP-synthase is constitutive.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Pseudomonas/enzimología , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Isoenzimas/metabolismo , Mutación , Fenilalanina/farmacología , Tirosina/farmacologíaAsunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Agrobacterium tumefaciens/metabolismo , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Aminoácidos Aromáticos/farmacología , Medios de Cultivo , Precursores Enzimáticos/farmacología , Represión Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Mutación , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Transaminasas/metabolismo , Triptófano Sintasa/metabolismo , Tirosina Transaminasa/metabolismoRESUMEN
Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and Tn5 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.
Asunto(s)
Conjugación Genética , Pseudomonas mendocina/genética , Mapeo Cromosómico , Cromosomas Bacterianos , Genoma Bacteriano , PlásmidosRESUMEN
Based on the results of matings with interrupted conjugation and analysis of marker joint inheritance frequencies, distances between 26 genetic determinants were estimated and a genetic map of Pseudomonas mendocina bacteria was constructed.
Asunto(s)
Genoma Bacteriano , Pseudomonas mendocina/genética , Conjugación GenéticaRESUMEN
A Tn10-containing variant of the pRK2013 plasmid, pRK2013-7, was used in the genetic analysis of Pseudomonas mendocina as chromosome-mobilizing inheritable factor that is able to integrate into the bacterial chromosome and transfer genetic markers with a frequency ranging from 3.2 x 10(-7) to 3.5 x 10(-3). The results of interrupted matings allowed localization of 10 genetic markers. This system of genetic analysis is suitable for P. mendocina mapping.
Asunto(s)
Mapeo Cromosómico/métodos , Genes Bacterianos , Genoma Bacteriano , Pseudomonas/genética , Conjugación GenéticaRESUMEN
It has been shown that under iron-limited conditions Pseudomonas putida M produces large amounts of the fluorescent pigment, pyoverdine Pm, which is a siderophore and exhibits antibacterial activity. The absorption spectrum for the pyoverdine Pm has two main peaks, at 230 nm and 400 nm, respectively. It was demonstrated that pyoverdine Pm molecule besides dihydroxyquinoline moiety has a peptide chain contain five amino acids: threonine, serine, lysine, hydroxyaspartic acid and N delta-hydroxyornithine in a molar ratio 3:2:1:1:1.
Asunto(s)
Oligopéptidos , Pigmentos Biológicos/química , Sideróforos/química , Aminoácidos/análisis , Colorantes Fluorescentes , Péptidos/química , Pigmentos Biológicos/biosíntesis , Sideróforos/biosíntesis , Espectrofotometría , Espectrofotometría UltravioletaRESUMEN
Dihydroorotate was shown to be a predecessor of deoxyquinoline nucleus of a fluorescing enzyme pyoverdin Pm in Pseudomonas putida M. The data was obtained in experiments with a set of Pyr- mutants with different steps of pyrimidine synthesis blocked. A scheme for deoxyquinoline nucleus of the enzyme including dihydroorotate participation is proposed.
Asunto(s)
Oligopéptidos , Pigmentos Biológicos/biosíntesis , Pirimidinas/metabolismo , Fluorescencia , Quinolinas/metabolismoRESUMEN
A new broad host range plasmid pM3 (IncP-9) was found in a facultative methylotrophic bacteria Pseudomonas putida and described. The pM3 plasmid is characterized by thermo-instability in Enterobacteriaceae family of bacteria at 36 degrees C or higher temperatures. It is also unable to be inherited as an autonomous element in the obligate methylotrophic bacteria Methylobacillus M75. The peculiarities of plasmid inheritance make possible to use it as a tool for genetic research, for instance, to construct the donor strains in Methylobacillus M75 able to mobilize the chromosomal genes for conjugational transfer in isogenic systems of crosses.
Asunto(s)
Bacterias Gramnegativas/genética , Plásmidos , Cromosomas Bacterianos , Conjugación Genética , ADN Bacteriano/genética , Genes Bacterianos , Especificidad de la EspecieRESUMEN
The activity of the first enzyme of aromatic path 3-deoxy-D-arabino-heptuloso-7-phosphate-synthase (DAHP-synthase) is regulated by retro-inhibition and is a subject of repression. Analysis of partially purified preparations of the enzyme has revealed three isoenzymes: DAHP-synthase-Tyr, DAHP-synthase-Trp and DAHP-synthase-Phe, each of them being regulated by a corresponding amino acid. DAHP-synthase-Phe is a dominant isoenzyme presenting 70% of the enzyme activity, 30% inhibition of which is possible by 7.0 mkM of phenylalanine. DAHP-synthase-Tyr and DAHP-synthase-Trp are minor isoenzymes (sharing 15% of enzyme activity each) and are controlled by tyrosine and tryptophane correspondingly. 50% of inhibition of activity is possible by adding 0.7 and 0.8 mkM of corresponding amino acid. Regulation of the enzyme synthesis was studied in the Trp-, Phe- and Tyr- mutants. The enzyme activity was registered under the conditions of limiting and surplus of each aromatic amino acid. The synthesis of DAHP-synthase in M. flagellatum KT is repressed by tryptophane and tyrosine decreasing the synthesis 18.8 and 15.6 fold.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Euryarchaeota/enzimología , Isoenzimas/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Aminoácidos/farmacología , Cromatografía DEAE-Celulosa , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metanol/metabolismo , MutaciónRESUMEN
Regulation of synthesis of 3-deoxy-D-arabinoheptulose-7-phosphate-synthase (DAHP-synthase) was analysed in 12 strains of Pseudomonas belonging to 10 species. Repression of synthesis of DAHP-synthase was registered in half of strains under study, this being controlled by tyrosin and phenilalanine in P. aeruginosa PAO1 and PAT 2152, P. fluorescens BKMB 896, P. acidovorans BKMB 1251 and P. testosteroni BKMB 1241 and by only phenilalanine in P. maltophilia BKMB 30. In the rest of cases (in P. putida AC29, P. stutzeri BKMB 148, P. mendocina BKMB 1299, P. marginata BKMB 1298, P. vesicularis BKMB 974 and P. fluorescens BKMB 35) the enzyme was synthesized constitutively.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Pseudomonas/enzimología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Fenilalanina/fisiología , Especificidad de la Especie , Tirosina/fisiologíaRESUMEN
Regulation of phenylalanine biosynthesis has been studied in the bacterium Methylobacillus M75 on the level of enzymes 3-deoxy-D-arabinoheptulose-7-phosphate-synthase, chorismatmutase, prephenatdehyrataze. The DAHP-synthase is shown to be synthesized constitutively and its activity is inhibited by all aromatic aminoacids and antranilate. The synthesis of chorismatmutase and prephenatdehydratase is repressed by tyrosine, the activity of the latter enzyme, besides that, is inhibited by phenylalanine, the effect of which is decreased in the presence of tyrosine.
Asunto(s)
Methylococcaceae/metabolismo , Fenilalanina/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Activación Enzimática , Methylococcaceae/enzimología , Methylococcaceae/genética , Mutación , Prefenato Deshidratasa/metabolismoRESUMEN
The transposon containing derivatives pMTF9 (Tn9), pMTF10 (Tn10) and pMTF59 (Tn5, Tn9) of the Pseudomonas sp. M conjugative plasmid pM3 demonstrating temperature-dependent instability in Erwinia cells incubated at 37 degrees C have been isolated. The obtained plasmids have been shown to be usable for transposon-mediated mutability in the bacterial cells of Erwinia generum incubated at 37 degrees C.
Asunto(s)
Elementos Transponibles de ADN , Erwinia/genética , Genes Bacterianos , Mutación , Plásmidos , Pseudomonas/genética , Cromosomas Bacterianos , Marcadores GenéticosRESUMEN
The synthesis of 3-deoxy-D-arabinoheptulosonate-7-phosphate-synthase has been shown to be repressed by tyrosine and phenylalanine in the cells of facultative methylotrophic bacteria Pseudomonas sp. M. Activity of the enzyme is subjected to allosteric inhibition by tyrosine, tryptophane, anthranylate and phenylpyruvate.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , Pseudomonas/enzimología , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , Regulación Alostérica , Aminoácidos/farmacología , Cromatografía DEAE-Celulosa , Mutación , Pseudomonas/genéticaRESUMEN
A method for isolation of Tyr- mutants of facultative methylotrophic bacteria Pseudomonas sp. M which possess two tyrosine synthesis pathways is presented. The method is based on the two-step blocking of the tyrosine synthesis: the first step of the supplementary pathway of synthesis from phenylalanine, the second being the main pathway from 4-hydroxyphenylpyruvate.
Asunto(s)
Mutación , Pseudomonas/genética , Tirosina/biosíntesis , Técnicas Bacteriológicas , Fenotipo , Pseudomonas/metabolismoRESUMEN
Regulatory 5-DL-methyltryptophan (5-MT)-resistant mutants of facultative methylotrophic Pseudomonas sp. M. were obtained. They are able to excrete tryptophan into the growth medium (60 to 300 g/ml). 5-MTR regulatory mutants are characterized by depression of trpE, trpD and trpC genes, which causes the production of intermediates of tryptophan biosynthesis and results in trpA and trpB genes induction as well as in two-fold activation of N-5-phosphoribosyl anthranilateisomerase (trpF gene product). Besides, all mutants demonstrate reduction of synthase feed-back inhibition about 4-11-fold. Together with tryptophan excretion, 5-MTR regulatory mutants are able to excrete tyrosine and unable to utilize this amino acid as the sole carbon source, which points to multiple nature of the selective effect of 5-MT.
Asunto(s)
Genes Reguladores , Mutación , Operón , Pseudomonas/genética , Triptófano/biosíntesis , Farmacorresistencia Microbiana , Pseudomonas/enzimología , Pseudomonas/metabolismo , Triptófano/análogos & derivados , Triptófano/genética , Triptófano/farmacologíaRESUMEN
Regulation of tryptophan biosynthesis of facultative methylotrophic Pseudomonas sp. M was studied. Repression of the trpE, trpD and trpC genes by tryptophan was demonstrated. It was also shown that the trpE and trpDC genes are derepressed noncoordinately. No regulation of the trpF gene product could be demonstrated, indicating that its synthesis is constitutive. The trpA and trpB genes are inducible by indol-3-glycerophosphate. Anthranilate synthase and tryptophan synthase were sensitive to the feedback inhibition. The tryptophan concentrations giving 50% inhibition were estimated to be 9 microM and 1 microM, respectively. Experimental evidence for activation of the N-5-phosphoribosyl anthranilate isomerase and for inhibition of the indol-3-glycerophosphate synthase by some tryptophan intermediates was obtained.
Asunto(s)
Genes Reguladores , Operón , Pseudomonas/genética , Triptófano/biosíntesis , Antranilato Sintasa/antagonistas & inhibidores , Activación Enzimática , Inducción Enzimática , Mutación , Pseudomonas/enzimología , Pseudomonas/metabolismo , Triptófano/genética , Triptófano Sintasa/antagonistas & inhibidoresRESUMEN
50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.
Asunto(s)
Conjugación Genética , Pseudomonas/genética , Factores R , Enzimas de Restricción del ADN , Metanol/metabolismo , Pseudomonas/metabolismoRESUMEN
A technique is proposed for the preparation of Pseudomonas putida protoplast-like structures by treating the cells with lysozyme in a hypertonic medium containing mono-and divalent cations. Pretreatment of the cells at the logarithmic growth phase with sucrose (0.5 M) for 90 min at the pH 7.9 to 8.0 is a prerequisite for the formation of 'protoplasts'. Under these conditions, the yield of 'protoplasts' reached 99.9% of the total cell number. The protoplast-like structures are capable of reversing into the original bacterial forms in a minimal hypertonic medium containing amino acids which are components of the cell wall. The level of reversion reaches 10% for certain strains.