RESUMO
Asparagine (Asn) is a powerful turn-inducer residue, with a large propensity to occupy the second position in the central region of ß-turns of proteins. The present work aims at investigating the role of a local anchoring between the Asn side chain and the main chain in this remarkable property. For this purpose, the H-bonding patterns of an asparagine residue in an isolated protein chain fragment forming a γ- or a ß-turn have been determined using IR/UV double resonance gas phase spectroscopy on laser-desorbed, jet-cooled short models in conjunction with relevant quantum chemistry calculations. These gas phase data provide evidence for an original double anchoring linking the Asn primary amide side chain (SC), which adopts a gauche+ rotameric form, to its main chain (MC) local environment. From both IR spectroscopic evidence (H-bond induced red shifts) and quantum chemistry, Asn SC is found to behave as a stronger H-bond acceptor than donor, resulting in stronger MCâSC H-bonds than SCâMC ones. These gas phase structural data, relevant to a hydrophobic environment, have been used as a reference to assess the anchoring taking place in high resolution crystallized proteins of the Protein Data Bank. This approach reveals that, when the SC adopts a gauche+ orientation, the stronger MCâSC bonds are preserved in many cases whereas the SCâMC bonds are always disrupted, in qualitative agreement with the gas phase ranking of these interactions. Most interestingly, when Asn occupies the second position of central part of a ß-turn (i.e., the very turn-inducer position), the MCâSC H-bonds are also disrupted and replaced by a water-mediated SC to MC anchoring. Owing to the specific features of the hydrated Asn side chain, we propose that it could be a turn precursor structure, able to facilitate turn formation in the early events of the folding process.
Assuntos
Asparagina/química , Peptídeos/química , Amidas/química , Gases/química , Ligação de Hidrogênio , Estrutura Secundária de Proteína , Teoria Quântica , Espectrofotometria InfravermelhoRESUMO
BACKGROUND: Hypersensitivity to somatic or visceral pain has been reported in numerous clinical conditions such as fibromyalgia or the irritable bowel syndrome, and general hypersensitivity has been proposed to be the underlying mechanism. However, cross-modal relationships especially between somatic and visceral pain have rarely been investigated even in healthy volunteers. Furthermore, psychological influences on pain have rarely been characterized across modalities. METHODS: Sixty-one healthy participants underwent testing of perception and pain thresholds for cutaneous thermode heat and cold, as well as for rectal balloon distension. Psychological testing for anxiety, depression, and pain experience (including catastrophizing and coping) as well as cardiac interoception was performed. Measurement quality and the correlations between the different modalities were examined. KEY RESULTS: Significant correlations existed between the perception thresholds for cold/heat (τB = -0.28, p = 0.002) and cold/distension (τB = -0.21, p = 0.03) and for the pain thresholds for cold/heat (r = -0.61, p < 0.001) and heat/distension (r = 0.33, p = 0.01). No association was found between pain thresholds and anxiety, depression, psychological experience with and processing of pain, or cardiac interoception. Retest reliabilities for pain measurements were satisfying after short intertrial intervals (all intraclass correlation coefficients >0.8), but less so after longer intervals. The individuals contributing to the respective correlations differ between measurements. CONCLUSIONS & INFERENCES: Moderate associations were found for pain thresholds across modalities. The strength of the associations and their stability over time warrants further investigation and might serve to increase the understanding of conditions affecting multiple pain modalities.
Assuntos
Percepção da Dor/fisiologia , Limiar da Dor/psicologia , Adulto , Feminino , Voluntários Saudáveis , Humanos , Masculino , Estimulação Física , Testes Psicológicos , Reto/fisiologia , Temperatura Cutânea/fisiologia , Adulto JovemRESUMO
BACKGROUND: Aplasia of the müllerian ducts leads to absence of the uterine corpus, uterine cervix, and upper (superior) vagina. Patients with müllerian aplasia (MA) often exhibit additional clinical features such as renal, vertebral and cardiac defects. A number of different syndromes have been associated with MA, and in most cases its aetiology remains poorly understood. OBJECTIVE AND METHODS: 14 syndromic patients with MA and 46,XX G-banded karyotype were screened for DNA copy number changes by approximately 1 Mb whole genome bacterial artificial chromosome (BAC) array based comparative genomic hybridisation (CGH). The detected alterations were validated by an independent method and further mapped by high resolution oligo-arrays. RESULTS: Submicroscopic genomic imbalances affecting the 1q21.1, 17q12, 22q11.21, and Xq21.31 chromosome regions were detected in four probands. Presence of the alterations in the normal mother of one patient suggests incomplete penetrance and/or variable expressivity. CONCLUSION: 4 of the 14 patients (29%) were found to have cryptic genomic alterations. The imbalances on 22q11.21 support recent findings by us and others that alterations in this chromosome region may result in impairment of müllerian duct development. The remaining imbalances indicate involvement of previously unknown chromosome regions in MA, and point specifically to LHX1 and KLHL4 as candidate genes.
Assuntos
Anormalidades Múltiplas/genética , Desequilíbrio Alélico , Aberrações Cromossômicas , Genitália Feminina/anormalidades , Ductos Paramesonéfricos/anormalidades , Adolescente , Adulto , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 22/genética , Cromossomos Humanos X/genética , Proteínas do Citoesqueleto/genética , Feminino , Dosagem de Genes , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Síndrome , Fatores de Transcrição , Útero/anormalidades , Vagina/anormalidades , Proteínas Wnt/genética , Proteína Wnt4RESUMO
gamma-Turn, the shortest secondary structure of peptides, exists as two helical forms gamma(l) and gamma(d) of opposite handedness. The present gas phase study of capped l-Phe-Xxx peptides (Xxx = l-Ala, d-Ala or Aib: aminoisobutyric acid) provides a unique example of intramolecular chiral recognition of the gamma-turn helicity on Ala or Aib by the neighbouring residue Phe within the chain. With the chiral l- or d-Ala residues, the presence of a side-chain operates a discrimination between the two helical forms: one of them is widely favoured over the other (gamma(l) or gamma(d), respectively). This enables us to validate and calibrate the recognition capabilities of the nearby l-Phe residue. The discriminating interactions have been precisely characterized from their spectroscopic UV and IR signatures and identified by comparison with quantum chemistry calculations. Then, in the case of the non-chiral residue Aib, the two helical forms of the gamma-turn, which are simultaneously observed in the jet, have been discriminated and assigned by comparison with the chiral residues. The relative abundances of the diastereomeric forms l-Phe-Aib(gamma(l)) and l-Phe-Aib(gamma(d)) enable us to determine the most efficient recognition configuration.
Assuntos
Aminoácidos/química , Peptídeos/química , Ligação de Hidrogênio , Estrutura Secundária de Proteína , Análise EspectralRESUMO
Lanthanides complexes are widely used as contrast agents in magnetic resonance imaging (MRI) and are involved in many fields such as organic synthesis, catalysis, and nuclear waste management. The complexation of the ion by the solvent or an organic ligand and the resulting properties (for example the relaxivity in MRI) are mainly governed by the structure and dynamics of the coordination shells. All of the MD approaches already carried out for the lanthanide(III) hydration failed due to the lack of accurate representation of many-body effects. We present the first molecular dynamics simulation including these effects that accounts for the experimental results from a structural and dynamic (water exchange rate) point of view.
RESUMO
AIMS: A possibility for the complementation of both ortho- and meta-cleavage pathway for chlorocatechols in one strain and its impact on degradation of chlorobenzoates accumulated during degradation of polychlorinated biphenyls was investigated. METHODS AND RESULTS: Genes responsible for ortho-cleavage of chlorocatechols were subcloned into two biphenyl degraders and the activities of chlorocatechol dioxygenases responsible for ortho- and meta-cleavage in these hybrid strains were monitored spectrophotometrically and also electrochemically by ion-selective electrode. CONCLUSIONS: While strain Pseudomonas fluorescens S12/C apparently gained metabolic advantage from this gene manipulation, strain Burkholderia cepacia P166/C did not express better degradation features in comparison with the parental strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach has the potential to enhance chlorocatechol metabolism in selected biphenyl degraders.
Assuntos
Catecóis/metabolismo , Clorobenzoatos/metabolismo , Bifenilos Policlorados/metabolismo , Pseudomonas fluorescens/genética , Biodegradação Ambiental , Burkholderia cepacia/genética , Burkholderia cepacia/metabolismo , Cloretos/análise , DNA Bacteriano , Eletroquímica , Engenharia Genética , Pseudomonas fluorescens/metabolismo , EspectrofotometriaRESUMO
Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contaminated by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strains Pseudomonas alcaligenes KP2 and P. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degraders Burkholderia cepacia strain LB400 and Ralstonia eutropha strain H850. The strain KP12 employs the same meta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degrader Pseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.
Assuntos
Bifenilos Policlorados/metabolismo , Pseudomonas/metabolismo , Microbiologia do Solo , Biodegradação Ambiental , Southern Blotting , Burkholderia cepacia/isolamento & purificação , Burkholderia cepacia/metabolismo , Cupriavidus necator/isolamento & purificação , Cupriavidus necator/metabolismo , República Tcheca , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Poluentes do Solo/metabolismo , Poluentes do Solo/toxicidadeRESUMO
We present a model potential for studying M(q+)(H(2)O)(n=1,9) clusters where M stands for either Na(+), Cs(+), Ca(2+), Ba(2+), or La(3+). The potential energy surfaces (PES) are explored by the Monte Carlo growth method. The results for the most significant equilibrium structures of the PES as well as for energetics are favorably compared to the best ab initio calculations found in the literature and to experimental results. Most of these complexes have a different coordination number in cluster compared to experimental results in solution or solid phase. An interpretation of the coordination number in clusters is given. In order to well describe the transition between the first hydration sphere and the second one we show that an autocoherent treatment of the electric field is necessary to correctly deal with polarization effects. We also explore the influence of the cation properties (charge, size, and polarizability) on both structures and coordination number in clusters, as well as the meaning of the second hydration sphere. Such an approach shows that the leading term in the interaction energy for a molecule in the second hydration sphere is an electrostatic attraction to the cation and not a hydrogen bond with the water molecules in the first hydration sphere.
RESUMO
Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the "upper" pathway are then mineralized by either ortho- or meta-ring cleavage ("lower" pathway). BTEX are relatively water-soluble and therefore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.
Assuntos
Bactérias Aeróbias/metabolismo , Hidrocarbonetos/metabolismo , Bactérias Aeróbias/enzimologia , Benzeno/metabolismo , Derivados de Benzeno/metabolismo , Biodegradação Ambiental , Hidrocarbonetos/química , Tolueno/metabolismo , Xilenos/metabolismoRESUMO
Microarrays of oligonucleotides or cDNAs can be used to establish the expression profiles of numerous genes in a single experiment. We have established a microarray platform to identify genes in a number of different pathological conditions, particularly those with an inflammation component. This platform utilised the output of an eosinophil sequencing project in which 1069 sequences were identified that were not represented in the public domain. An eosinophil model cell line, AML14.3D10, was used to investigate cell adhesion. The transcription profile of adhered and non-adhered AML 14.3D10 cells was shown to be both technically and biologically reproducible. A number of genes were found differentially expressed in the adhered vs. non-adhered populations. In the adhered population, the expression of these genes was restricted compared to brain, lung, kidney and especially bone marrow. However, the differentially regulated genes were not among those genes most restricted to eosinophils. We discuss the implications of transcription profiling on gene annotation and its potential utility for the identification of targets for drug intervention.
Assuntos
Adesão Celular/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Eosinófilos/citologia , Eosinófilos/fisiologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/estatística & dados numéricos , Biblioteca Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
Two polychlorinated biphenyl-contaminated sites in the Czech Republic, a soil at Zamberk and a sediment sludge at Milevsko, were screened for the presence of chlorobenzoate degraders. Sixteen different chlorobenzoate degraders were isolated from the soil compared with only three strains isolated from the sediment. From these strains, only four soil degraders and one strain isolated from the sediment, respectively, were shown to possess a complete chlorobenzoate (CB) pathway. Bacteria isolated from the soil have expressed more flexibility for CB degradation, namely in the case of ortho-chlorinated benzoates. They all possessed large plasmids, the restriction patterns of which were compared. Plasmids in Pseudomonas sp. A7, A8, A18 and A19, respectively, were cured and found to encode at least part of the metabolic pathway involved in the growth on ortho-chlorinated benzoates.
Assuntos
Clorobenzoatos/metabolismo , Dioxigenases , Bifenilos Policlorados , Pseudomonas/metabolismo , Microbiologia do Solo , Poluentes do Solo , Biodegradação Ambiental , Catecol 1,2-Dioxigenase , República Tcheca , DNA Bacteriano , Oxigenases/análise , Plasmídeos , Pseudomonas/isolamento & purificaçãoRESUMO
The relationship between parenting practices and behavior problems in very young children was investigated via the Parent Behavior Checklist (PBC; Fox, 1994). Results indicated that parents' use of verbal and corporal punishment was the strongest predictor of reported behavior problems, accounting for 20% of overall variance and 13% of unique variance. Results for parental nurturing and expectations were mixed and suggest an indirect effect. Several demographic variables were also predictive of behavior problems. Implications for Patterson's (1986) model of the development of antisocial behavior and for the use of parent assessment as a screening tool for preventive interventions are discussed.
Assuntos
Transtornos do Comportamento Infantil/psicologia , Educação Infantil , Relações Mãe-Filho , Punição , Fatores Etários , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Psicologia da CriançaRESUMO
In this paper we present the entire genomic sequence as well as the cDNA sequence of two new human genes encoding the gamma subunit of the NAD(+)-dependent isocitrate dehydrogenase (H-IDH gamma) and the translocon-associated protein delta subunit (TRAP delta). These genes are located on region q28 of the human X chromosome, approximately 70 kb telomeric to the adrenoleukodystrophy locus (ALD). The sequences of the transcripts of both genes were obtained by searching the EST database with genomic data. Identified ESTs were completely sequenced and assembled to cDNAs comprising the entire coding region. For IDH gamma, several EST clones indicate differential splicing. IDH gamma and TRAP delta are arranged in a compact head to head manner. The nontranscribed intergenic region represents only 133 bp and is embedded in a CpG island. The CpG island obviously functions as a bidirectional promoter to initiate the transcription of both functionally unrelated genes with quite distinct expression patterns. This exceptional gene arrangement prompted us to clone and sequence genomic DNA fragments containing the homologous intergenic regions of rat and mouse. We show that in both species this area is similarly compact and represents less than 249 bp in rat and not more than 164 bp in mouse. In both cases this intergenic region is embedded in a CpG island and is highly conserved with nucleotide identity values ranging from 70.1% between human and rat to 92.6% between mouse and rat.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Isocitrato Desidrogenase/genética , Glicoproteínas de Membrana , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/genética , Cromossomo X/genética , Adrenoleucodistrofia/genética , Animais , Sequência de Bases , Clonagem Molecular , Ilhas de CpG/genética , Primers do DNA , Bases de Dados Factuais , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Regiões Promotoras Genéticas , Splicing de RNA/genética , Ratos , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
While the addictive potential of Internet usage is a topic that has attracted a great deal of attention, as yet little research has addressed this topic. Preliminary data from the Internet Usage Survey shows that most of the 563 users reported instances of Internet use interfering with other aspects of their lives, most commonly problems with managing time. A subgroup of users endorsed multiple usage-related problems, including several similar to those found in addictions. Younger users tended to have experienced more problems.
Assuntos
Atitude Frente aos Computadores , Comportamento Aditivo/epidemiologia , Redes de Comunicação de Computadores , Adulto , Comportamento Aditivo/psicologia , Comparação Transcultural , Feminino , Inquéritos Epidemiológicos , Humanos , Masculino , Inventário de PersonalidadeRESUMO
During the course of a large-scale sequencing project in Xq28, a human creatine transporter (CRTR) gene was discovered. The gene is located approximately 36 kb centromeric to ALD. The gene contains 13 exons and spans about 8.5 kb of genomic DNA. Since the creatine transporter has a prominent function in muscular physiology, it is a candidate gene for Barth syndrome and infantile cardiomyopathy mapped to Xq28.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Cromossomo X , Sequência de Bases , Cardiomiopatias/genética , Proteínas de Transporte/biossíntese , Centrômero , Mapeamento Cromossômico , Fosfatos de Dinucleosídeos , Repetições de Dinucleotídeos , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Doenças Neuromusculares/genética , Oligodesoxirribonucleotídeos , TATA BoxRESUMO
OBJECTIVE: The long-term effects of a combination of clozapine and psychosocial treatment were evaluated in a sample of treatment-refractory state hospital patients with schizophrenia. METHODS: A repeated-measure design was used. Thirty-one patients with schizophrenia received both clozapine and an enhanced psychosocial treatment program. Data were collected at baseline and at one-year, two-year, and three-year follow-ups. Psychiatric symptoms, cognitive functioning, dyskinetic movements, and discharge rate were evaluated. RESULTS: Significant reductions in psychiatric symptoms and improvement in cognitive functioning were found. Differences in the pattern of reductions in positive and negative symptoms over the course of the study were noted. The majority of subjects improved sufficiently to be discharged. CONCLUSIONS: Clozapine, when combined with psychosocial treatment, is effective for treatment of patients with schizophrenia who are not responsive to other medications.
Assuntos
Antipsicóticos/administração & dosagem , Clozapina/administração & dosagem , Transtornos Cognitivos/reabilitação , Esquizofrenia/reabilitação , Psicologia do Esquizofrênico , Terapia Socioambiental , Adulto , Antipsicóticos/efeitos adversos , Clozapina/efeitos adversos , Transtornos Cognitivos/psicologia , Feminino , Seguimentos , Humanos , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Escalas de Graduação Psiquiátrica , Resultado do TratamentoRESUMO
Genetic construction of recombinant strains with expanded degradative abilities may be useful for bioremedation of recalcitrant compounds, such as polychlorinated biphenyls (PCBs). Some degradative genes have been found either on conjugative plasmids or on transposons, which would facilitate their genetic transfer. The catabolic pathway for the total degradation of PCBs is encoded by two different sets of genes that are not normally found in the same organism. The bphABCD genes normally reside on the chromosome and encode for the four enzymes involved in the production of benzoate and chlorobenzoates from the respective catabolism of biphenyl and chlorobiphenyls. The genes encoding for chlorobenzoate catabolism have been found on both plasmids and the chromosome, often in association with transposable elements. Ring fission of chlorobiphenyls and chlorobenzoates involves the meta-fission pathway (3-phenylcatechol 2,3-dioxygenase) and the ortho-fission pathway (chlorocatechol 1,2-dioxygenase), respectively. As the catecholic intermediates of both pathways are frequently inhibitory to each other, incompatibilities result. Presently, all hybrid strains constructed by in vivo matings metabolize simple chlorobiphenyls through complementary pathways by comprising the bph, benzoate, and chlorocatechol genes of parental strains. No strains have yet been verified which are able to utilize PCBs having at least one chlorine on each ring as growth substrates. The possible incompatibilities of hybrid pathways are evaluated with respect to product toxicity, and the efficiency of both in vivo and in vitro genetic methods for the construction of recombinant strains able to degrade PCBs is discussed.
Assuntos
Bactérias/genética , Bifenilos Policlorados/metabolismo , Bactérias/metabolismo , Biodegradação Ambiental , Clorobenzoatos/metabolismo , Engenharia GenéticaRESUMO
Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Kröckel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CC1 and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 mug ml and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction of P. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use of meta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.
RESUMO
Pseudomonas putida P111 is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates. The involvement of two separate dioxygenases was noted from data on plasmid profiles and DNA hybridization. The benzoate dioxygenase, which converts 3-chlorobenzoate (3-CB), 4-CB, and benzoate to the corresponding catechols via reduction of a dihydrodiol, was shown to be chromosomally coded. The chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to the corresponding catechols without the need of a functional dioldehydrogenase was shown to be encoded on plasmid pPB111 (75 kb). Cured strains were unable to utilize ortho-chlorobenzoates for growth. DNA hybridization data indicated that catabolism of the corresponding chlorocatechols was coded on chromosomal genes. Maintenance of plasmid pPB111 was dependent on the presence of ortho-chlorobenzoates in the growth media. A unique variant of P111 (P111D), able to grow on 3,5-dichlorobenzoate (3,5-DCB), was obtained by continuous subculturing from media containing progressively lower and higher concentrations of 3-CB and 3,5-DCB, respectively. The low frequency of segregants able to grow on 2,5-DCB, 2,3-DCB, and 2,3, 5-trichlorobenzoate was evident by lag periods greater than 200 h. Continued subculture on 3,5-DCB resulted in the formation of new plasmid pPH111 (120 kb), which was homologous to pPB111. A probe from the clc operon, which encodes for the chlorocatechol pathway, hybridized to plasmid pPH111 and to the chromosome of the wild-type strain P111 but not to its plasmid pPB111 nor to the chromosome of strain P111A, which had lost the ability to utilize chlorobenzoates.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Clorobenzoatos/metabolismo , Pseudomonas putida/metabolismo , Biodegradação Ambiental , Genes Bacterianos , Variação Genética , Óperon , Fenótipo , Plasmídeos , Pseudomonas putida/genética , Pseudomonas putida/crescimento & desenvolvimentoRESUMO
To develop a host-vector system for Methylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, the E. coli--Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Apr, Tcr) was constructed by joining the E. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium, Methylobacterium sp. strain M17. Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidless Methylobacterium sp. strain R2b. The stability of pWUBR in Tcr methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90% in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.