RESUMEN
A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Ácido Aminoetilfosfónico/metabolismo , Ácido Fosfonoacético/metabolismo , Rhodobacteraceae , Acetaldehído/análogos & derivados , Acetaldehído/metabolismo , Organismos Acuáticos/enzimología , Organismos Acuáticos/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Concentración de Iones de Hidrógeno , NADP/metabolismo , Fósforo/metabolismo , Rhodobacteraceae/enzimología , Rhodobacteraceae/crecimiento & desarrollo , Rhodobacteraceae/aislamiento & purificación , Especificidad por Sustrato , Temperatura , Transaminasas/metabolismoRESUMEN
Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.
Asunto(s)
Fosfatos/metabolismo , Polifosfatos/metabolismo , Aguas del Alcantarillado/microbiología , Sulfitos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Biodegradación Ambiental/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fosfatos/aislamiento & purificación , Polifosfatos/aislamiento & purificaciónRESUMEN
The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5' ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.
Asunto(s)
Operón/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Pseudomonas fluorescens/enzimología , Fosfatasa Alcalina , Secuencia de Aminoácidos , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Monoéster Fosfórico Hidrolasas/química , Propionatos/metabolismo , Pseudomonas fluorescens/genética , Análisis de Secuencia de ADN , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The phnA gene encoding a novel carbon-phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.
Asunto(s)
Escherichia coli/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Pseudomonas fluorescens/enzimología , Pseudomonas putida/genética , Fosfatasa Alcalina , Secuencia de Aminoácidos , Secuencia de Bases , Carbono/metabolismo , Clonación Molecular , Datos de Secuencia Molecular , Fósforo/metabolismo , Pseudomonas fluorescens/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de SecuenciaRESUMEN
Cryptic plasmids were found in Rhodococcus rhodochrous NCIMB13064 derivatives which had lost the ability to utilize short-chain 1-chloroalkanes (chain length C3-C10) and had acquired the ability to degrade naphthalene. The reversions of these derivatives to the original phenotype were accompanied by the loss of the cryptic plasmids. The 4969-bp pKA22 plasmid was cloned in Escherichia coli and sequenced. This plasmid encodes a putative 33,200-Da protein which contains motifs typical of theta replicase proteins and shows a high degree of similarity to a putative theta replicase from Brevibacterium linens plasmid pRBL1 and to a putative protein encoded by ORF1 of the plasmid pAL5000 from Mycobacterium fortuitum. Two sets of long direct repeats were found in pKA22 which may be involved in the replication of the plasmid and recombination processes.
Asunto(s)
Naftalenos/metabolismo , Plásmidos/genética , Rhodococcus/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Biodegradación Ambiental , Clonación Molecular , Replicación del ADN/genética , ADN Bacteriano/genética , ADN Polimerasa Dirigida por ADN/genética , Hidrocarburos Clorados/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fenotipo , Recombinación Genética/genética , Secuencias Repetitivas de Ácidos Nucleicos , Rhodococcus/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C3-C8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.
Asunto(s)
Hidrocarburos Clorados/metabolismo , Naftalenos/metabolismo , Rhodococcus/aislamiento & purificación , Rhodococcus/metabolismo , Microbiología del Suelo , Ácido Pirúvico/metabolismoRESUMEN
Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C3 to C9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C12 to C18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.
Asunto(s)
Genes Bacterianos , Hidrocarburos Clorados/metabolismo , Plásmidos , Rhodococcus/genética , Rhodococcus/metabolismo , Biotransformación , Cruzamientos Genéticos , Marcadores Genéticos , Mitomicina/farmacología , Fenotipo , Plásmidos/aislamiento & purificación , Rhodococcus/efectos de los fármacos , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho-pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentistic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.
Asunto(s)
Naftalenos/metabolismo , Plásmidos/fisiología , Pseudomonas putida/crecimiento & desarrollo , Conjugación Genética/fisiología , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMEN
The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid. The plasmid may be transferred by conjugation into P. aeruginosa PAO2175. It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol. The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system. The resulting pBS110 plasmid was transferred into P. putida, which results in utilization of 3-chlorobenzoate by transconjugants.
Asunto(s)
Clorobenzoatos/metabolismo , Genes Bacterianos , Pseudomonas putida/genética , Biodegradación Ambiental , Clonación Molecular , Conjugación Genética , ADN Bacteriano/genética , Escherichia coli/metabolismo , Plásmidos , Mapeo RestrictivoRESUMEN
Most of the known naphthalene biodegradation plasmids determine the process of naphthalene degradation via salicylate and catechol using the meta pathway of catechol degradation. However, Pseudomonas putida strains with plasmids pBS2, pBS216, pBS217 and NPL-1 exert no activity of the enzymes involved in the meta pathway of catechol degradation. When 2-methylnaphthalene was added to the medium as a sole carbon source, mutants growing on this compound were isolated in the strains with the studied plasmids. Plasmid localization of the mutations was established using conjugation transfer as well as by obtaining spontaneous variants that had lost the ability to grow on 2-methylnaphthalene; the respective plasmid mutants were referred to as pBS101, pBS102, pBS103 and pBS105. The strains with the mutant plasmids were tested for the activity of the key enzymes involved in naphthalene catabolism and the activity of catechol-2,3-dioxygenase was found. The data allow one to arrive at the conclusion that plasmids pBS2, pBS216, pBS217 and NPL-1 contain silent genes for the meta pathway of catechol degradation, which are activated by the respective mutations.
Asunto(s)
Catecoles/metabolismo , Mutación , Naftalenos/farmacocinética , Plásmidos , Pseudomonas/genética , Biodegradación Ambiental , Genes Bacterianos , Oxidación-Reducción , Pseudomonas/metabolismoAsunto(s)
Catecoles/metabolismo , Dioxigenasas , Genes Bacterianos , Naftalenos/metabolismo , Plásmidos , Pseudomonas/genética , Biodegradación Ambiental , Catecol 1,2-Dioxigenasa , Catecol 2,3-Dioxigenasa , Oxidación-Reducción , Oxigenasas/metabolismo , Fenotipo , Pseudomonas/crecimiento & desarrollo , Pseudomonas/metabolismoAsunto(s)
Dicofol/farmacocinética , Insecticidas/farmacocinética , Naftalenos/farmacocinética , Oxidorreductasas/metabolismo , Plásmidos , Pseudomonas aeruginosa/enzimología , Biodegradación Ambiental , Oxidación-Reducción , Fenotipo , Pseudomonas aeruginosa/genética , Microbiología del Suelo , Contaminantes del SueloRESUMEN
The paper contains the data on the structure of NPL-1 plasmid controlling naphthalene biodegradation. The plasmid which pertains to the P-9 incompatibility group is transferrable conjugatively and is maintained stably within a wide range of gram-negative bacteria. The analysis of mutants and transposon derivatives of the plasmid made it possible to localize nah-genes in a DNA fragment, 23 kb in size. An inverted DNA segment of 4.2 kb was discovered which may participate in the regulation of nah-genes expression. The other peculiar features of NPL-1 were found distinguishing it from NAH and NAH7 plasmids described in literature.
Asunto(s)
Naftalenos/metabolismo , Plásmidos , Pseudomonas/genética , Biodegradación Ambiental , Enzimas de Restricción del ADN , Elementos Transponibles de ADN , Marcadores Genéticos , Mutación , Oxidación-Reducción , Pseudomonas/metabolismo , Especificidad de la EspecieRESUMEN
The stability of biodegradation plasmids NPL-1 and NPL-41, which control the synthesis of enzymes for naphthalene oxidation to salicylate, was studied in Pseudomonas putida BSA under the conditions of its continuous cultivation with limitation in glucose or salicylate in the chemostat regime and without limitation in the pH-stat regime. Plasmid NPL-1, which controls the inducible synthesis of naphthalene oxygenase, is stable in the population of P. putida cells under the conditions of continuous cultivation on glucose, but is not stable in the course of cultivation on salicylate, an inductor of the naphthalene oxygenase synthesis. Plasmid NPL-41, which controls the constitutive synthesis of naphthalene oxygenase, is not stable in the population of P. putida cells under the conditions of continuous cultivation on glucose. The operation of genes, which control the oxidation of naphthalene to salicylate (nah), makes plasmids NPL-1 and NPL-41 unstable under the conditions of continuous cultivation in the absence of naphthalene from the medium, i.e. under the conditions when the expression of these genes is not necessary. In that case, cells containing plasmids with a deletion of nah-genes as well as cells without plasmids appear in the population of P. putida, which causes a decline in its futile energy and metabolic processes.