Biomedical subjects

J L Ramos

Publications and source records attributed to J L Ramos.

At least 109 records · Page 6Linked to original sources

PubMed1993

The XylS/AraC family of regulators.

At least twenty-seven proteins belong to the XylS/AraC family of prokaryote transcriptional regulators. All members of this family except CelD and TetD are positive transcriptional factors. Three subgroups were distinguished within the family in accordance with the Needleman and Wunsch algorithm. Multiple alignment of these proteins revealed that they shared a high degree of sequence homology at their C-terminal end, where a characteristic conserved motif, whose consensus sequence is I-DIA--GF-S--YF--F---G-TPS--R (where - means any aminoacid), was found. Within the homologous C-terminal region, but outside the above consensus motif, a putative DNA-binding domain organized as a helix-turn-helix motif was located in all regulators. For regulators recognizing chemical signals, the non-homologous N-terminal region of these regulators is presumed to contain binding sites for activator molecules that confer specificity.

Amino Acid Sequence↗

PubMed1993

Physical organization of the upper pathway operon promoter of the Pseudomonas TOL plasmid. Sequence and positional requirements for XylR-dependent activation of transcription.

The upper pathway operon of the Pseudomonas putida TOL plasmid belongs to the -12/-24 class of promoters. These promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for RNA polymerase/sigma 54 binding, the -55/-67 region for IHF protein binding, and the -130(UAS2)/-170(UAS1) region, where two sites for XylR binding are located. The XylR-protected G residues located at -131, -139, -160 and -169 were replaced with As, and the activity of the mutant promoters was assayed after fusion to a promoterless lacZ gene. The mutation (G(-169)-->A) resulted in a 50% decrease in expression from the promoter (Pu), whereas the other three changes had no significant effect. The XylR recognition sequence UAS2 has a perfect inverted repeat (5'-ATTTN4-AAAT-3') while UAS1 shows two mismatches (5'-CCTTN4AAAT-3'). The two Cs (located at -172 and -173), which interrupt the inverted repeat, were changed as follows: C(-172)-->T; C(-173)-->A, CC(-172, -173)-->AT. Transcription activation from the mutant promoters was measured as beta-galactosidase activity after fusion to lacZ; the better the palindromic sequence, the higher the rate of transcription from Pu, with increases in activity of up to 50%. The introduction of one or two full helix turns between the IHF and the XylR binding sites did not significantly affect transcription from Pu; however, the insertion of three helix turns resulted in a drop of 90% in the activity. The non-permissive effect of insertion of three full helix turns between the IHF and XylR binding sites was not evident in an IHF- background.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1993

Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways.

TOL plasmid pWW0 of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the metacleavage pathway operon encodes the enzymes for the further oxidation of these compounds to Krebs cycle intermediates. Their expression is controlled by the gene products of two divergently transcribed regulatory genes, xyIR and xyIS. The XyIR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XyIR stimulates transcription from the xyIS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are sigma 54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XyIR and IHF in the Pu promoter and for XyIR in the Ps promoters have been defined. The XyIS protein, which belongs to the AraC family of regulators, stimulates transcription from the meta-cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and DNA-binding region of XyIS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the meta-cleavage pathway operon is also induced by xylene-activated XyIR protein via a cascade regulatory system in which this protein, in combination with sigma 54, stimulates the expression from the xyIS promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1993

A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene.

A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA1-04/03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In bacteria bearing two copies of the killing cassette, the rate of appearance of mutants resistant to killing decreased to as low as 10(-8) per cell per generation.

Benzoates↗

PubMed1993

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.

Hybridization, Genetic↗

PubMed1993

Suicidal genetic elements and their use in biological containment of bacteria.

The potential risks of unintentional releases of genetically modified organisms, and the lack of predictable behavior of these in the environment, are the subject of considerable concern. This concern is accentuated in connection with the next phase of gene technology comprising deliberate releases. The possibilities of reducing such potential risks and increasing the predictability of the organisms are discussed for genetically engineered bacteria. Different approaches towards designing disabled strains without seriously reducing their beneficial effects are presented. Principally two types of strain design are discussed: actively contained bacteria based on the introduction of controlled suicide systems, and passively contained strains based on genetic interference with their survival under environmental-stress conditions.

Bacteria↗

PubMed1993

Mineralization of p-methyl-14C-benzoate in soils by Pseudomonas putida (pWW0).

Pseudomonas putida bearing the archetypal TOL plasmid pWW0 metabolizes p-methylbenzoate through a meta-cleavage pathway. In complex environments such as soils that are relatively rich in organic matter, we observed metabolism of p-methyl-14C-benzoate, which could be monitored as 14CO2 evolution from the labelled alkylaromatic. Linear 14CO2 evolution in soils took place for at least a month, although efficient mineralization of the alkylaromatic required appropriate mass transfer to allow the bacterial cell and target chemical to remain in close contact.

Benzoates↗

PubMed1992

Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid.

The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.

Alleles↗

PubMed1992

Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid.

The xylR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters. Transcription from the xylS gene promoter, Ps, is positively regulated by effector-activated XylR protein and requires the specific RNA polymerase sigma 54 subunit (RpoN). Deletions and point mutations in the Ps upstream region localized the site of XylR interaction to the region between -133 bp and -207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XylR in the XylR-regulated 'upper' catabolic pathway promoter, Pu. Gel retardation experiments showed binding of IHF to the Ps promoter region. Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between -35 bp and -47 bp, Ps proximal site) and further upstream overlapping the XylR recognition sequence (Ps distal site). In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between -141 bp and -153 bp on the lower strand). Expression from Ps, either under inducing or non-inducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma 54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.

Amino Acid Sequence↗

PubMed1992

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1992

Nitroaromatics Are Substrates for the TOL Plasmid Upper-Pathway Enzymes.

Expression of the xylMA genes encoding for toluene monoxygenase from the lactose promoter in a broad-host-range plasmid allows the oxidation of toluene and m- and p-nitrotoluene to their corresponding benzyl alcohols and benzaldehydes in Pseudomonas putida and Escherichia coli. Benzyl alcohols accumulate until reaching a concentration of about 80 muM, while benzaldehydes accumulate steadily with time for at least 24 h. TOL-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase recognize m- and p-nitro-substituted compounds as substrates. In contrast, the XylR protein, which regulates the TOL plasmid-encoded upper-pathway operon, does not recognize nitro-substituted toluenes as effectors.

Journal Article↗

PubMed1992

Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440.

Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida (pWW0) released in unsterile lake mesocosms.

Antibodies, Monoclonal↗

PubMed1992

Seizure recurrence in infants with neonatal convulsions. A follow-up study.

Twenty three infants with neonatal seizures were followed prospectively to a mean age of 11 months. Only 2 were pre-term and birth weight ranged from 1700 to 4230 grams, with 17 male and 6 female infants. Hypoxic-ischemic encephalopathy was the most common etiology (82.6%). Focal clonic convulsions were the predominant seizure type, present in 7/16 infants in which the seizure type could be identified. All infants had a neurological examination and EEG, and 18 had a cranial ultrasonography performed at the follow-up. Anticonvulsant medication was discontinued, if follow-up EEG and neurological examination were normal. At the follow-up, seizure recurrence was observed in 7/23 (30%) infants. Abnormal EEG, neurological examination and cranial ultrasonography were statistically correlated with seizure recurrence. We conclude that infants with neonatal seizures can remain free of anticonvulsant medication provided they have normal neurological examination, EEG and cranial ultrasonography.

Echoencephalography↗

PubMed1992

Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics.

Pseudomonas sp. FR1 (pFRC20P) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylXYZL and xylS genes from the TOL plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from Alcaligenes eutrophus on pFRC20P. The survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil. The recombinant information was stable in bacteria introduced in soils and conferred selective advantage to the host bacterium when the soils were amended with low amounts of p-methylbenzoate. However, relatively high amounts of this chemical significantly reduced survival of the bacterium. Survival was more seriously affected by chlorobenzoates than by methylbenzoates. Evolution of 14CO2 from p-methyl-14C-benzoate in soils confirmed that Pseudomonas sp. FR1 (pFRC20P) functions in a complex system in accordance with its design. Survival of Pseudomonas sp. FR1 (pFRC20P) was better at 4 degrees C and 25 degrees C than at 37 degrees C. On plates, the pFRC20P plasmid was mobilized by helper plasmids to P. putida at a frequency of about 10(-6) transconjugants per recipient. However, no transfer of recombinant DNA integrated on the chromosome was found, nor was lateral transfer of the recombinant DNA borne by Pseudomonas sp. FR1 (pFRC20P) detected in soils.

Benzoates↗

PubMed1991

Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein.

Expression of the Pseudomonas putida TOL plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class. Stimulation of transcription from this promoter is positively controlled by the effector-activated XylR protein and requires a form of RNA-polymerase holoenzyme containing the RpoN-encoded sigma factor, sigma 54. XylR-dependent stimulation of transcription from the Pseudomonas TOL upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate footprinting. Two upstream activator sequences were identified in the -160 (UAS1) and -130 (UAS2) regions. Deletion of these two regions abolished transcription activation, although conservation of the UAS2 element alone allowed limited transcription stimulation. Separation of UAS1 from UAS2 by half a turn or a full turn significantly reduced XylR stimulation of transcription from the upper pathway operon promoter. An inverted repeated ATTTGN2CAAAT (where N is any nucleoside), which most likely represented the XylR recognition sequence, was identified. Binding of XylR was observed in vivo in the absence of effector, but changes in the binding pattern were induced in the presence of m-methylbenzyl alcohol, a XylR effector. In vivo footprinting analysis revealed that changes in the methylation pattern of G and T also occurred in the -50 to -90 region, which is probably occupied by integration host factor (IHF) protein. IHF was required for maximal expression from the TOL upper pathway operon promoter in Escherichia coli. Separation of the IHF site from UAS2 by a full helix turn did not significantly affect stimulation of transcription, which is consistent with this region playing a conformational role, rather than a regulatory one, in promoter function.

Autoradiography↗

PubMed1991

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid.

Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.

Aminobutyrates↗

PubMed1991

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.

Anti-Bacterial Agents↗

PubMed1991

Conditional-suicide containment system for bacteria which mineralize aromatics.

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta-cleavage pathway operon (P(m)) and the lacI gene encoding Lac repressor plus xylS, coding for the positive regulator of P(m). The other element carries a fusion between the P(tac) promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the P(tac)::gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.

Journal Article↗