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V de Lorenzo

Publications and source records attributed to V de Lorenzo.

At least 19 recordsLinked to original sources
PubMed2002

Deciphering the action of aromatic effectors on the prokaryotic enhancer-binding protein XylR: a structural model of its N-terminal domain.

The prokaryotic enhancer-binding protein XylR is the central regulator of the toluene degradation pathway in Pseudomonas species. Copious genetic and biochemical data indicate that the N-terminal domain of the protein (domain A) interacts directly with m-xylene, which renders the protein competent as a transcriptional activator. Single-site and shuffling mutants of XylR or homologues have been reported to change or expand their effector profiles. Here, we follow a fold recognition approach to generate three-dimensional models of the domain A of XylR and DmpR with the purpose of deciphering the molecular activity of this protein family. The model is based on the crystallographic data of the rat catechol O-methyltransferase, a typical alpha/beta fold, consisting of eight alpha-helices and seven beta-strands. The fold identification is supported by physico-chemical properties of conserved amino acids, distribution of residues characteristic of the sequence families and confrontation with experimental data. The model not only provides a rationale for understanding published experimental data, but also suggests the molecular mechanism of the activation step and is a potentially useful conceptual tool for designing regulators with predefined inducer specificities.

Amino Acid Sequence↗
PubMed2001

Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene.

In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.

Alcohol Oxidoreductases↗
PubMed2001

The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions.

A protein mixture containing two major components able to catalyze a beta-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract. N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant. The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence. The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified. Their activities were examined in vitro in beta-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis. HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins. This was reflected in the maintenance of in vivo activity of the final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN strains. However, hupB/hupN double mutants were not viable. It is suggested that the requirement for protein-facilitated DNA bending is met in P. putida by two independent proteins that ensure an adequate supply of an essential cellular activity.

Amino Acid Sequence↗
PubMed2001

Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity.

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and PM: Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the PM: promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

Bacterial Proteins↗
PubMed2001

Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway.

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.

Bacterial Proteins↗
PubMed2001

A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors.

To investigate the activation mechanism of the enhancer-binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled N-terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure. When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g. nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors. Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulators à la carte that are responsive to predetermined, natural or xenobiotic chemical species.

Bacterial Proteins↗
PubMed2001

New insights into the activation of o-xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates.

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the sigma54-dependent PToMO promoter and the sigma54-containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.

Adenosine Triphosphatases↗
PubMed2001

The limits to genomic predictions: role of sigma(N) in environmental stress survival of Pseudomonas putida.

Based on genomic data and on the phenotypes of an FlhF mutant of Pseudomonas putida, the alternative sigma factor sigma(N) (sigma(54)) has been proposed to play a key role in survival to various nutritional and environmental stresses in this bacterium. Quite in contrast, we show that unlike sigma(S) (sigma(38)) the loss of sigma(N) does not impair to any significant extent the ability of P. putida to survive long-term starvation. rpoN mutants (lacking sigma(N)) are indistinguishable from the wild-type with respect to solvent tolerance, resistance to heat shock or sensitivity to hydrogen peroxide. These data suggest that while sigma(N) is a key component of expression of alternative biodegradative pathways for unusual carbon sources (i.e. m-xylene or dimethylphenols), its loss does not compromise bacterial endurance to gross types of environmental stress. Moreover, these results point out the limitations, if not the deception, of genomic predictions when confronted with experimental data.

Journal Article↗
PubMed2001

Role of ptsO in carbon-mediated inhibition of the Pu promoter belonging to the pWW0 Pseudomonas putida plasmid.

An investigation was made into the role of the ptsO gene in carbon source inhibition of the Pu promoter belonging to the Pseudomonas putida upper TOL (toluene degradation) operon. ptsO is coexpressed with ptsN, the loss of which is known to render Pu unresponsive to glucose. Both ptsN and ptsO, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA(Ntr) and NPr, respectively, have been mapped adjacent to the rpoN gene of P. putida. The roles of these two genes in the responses of Pu to glucose were monitored by lacZ reporter technology with a P. putida strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking ptsO, Pu activity seemed to be inhibited even in the absence of glucose. A functional relationship with ptsN was revealed by the phenotype of a double ptsN ptsO mutant that was equivalent to the phenotype of a mutant with a single ptsN disruption. Moreover, phosphorylation of the product of ptsO seemed to be required for C inhibition of Pu, since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate ptsO revealed the presence of two open reading frames, designated ptsP and mtp, with the potential to encode PTS type I enzymes in P. putida. However, neither an insertion in ptsP nor an insertion in mtp resulted in a detectable change in inhibition of Pu by glucose. These results indicate that some PTS proteins have regulatory functions in P. putida that are independent of their recognized role in sugar transport in other bacteria.

Biological Transport, Active↗
PubMed2001

Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins.

We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

Amino Acid Sequence↗
PubMed2000

Evidence of an unusually long operator for the fur repressor in the aerobactin promoter of Escherichia coli.

Production of the siderophore aerobactin in Escherichia coli is transcriptionally metalloregulated through the iron-dependent binding of the Fur (ferric uptake regulator) to a large region (>100 base pairs) within the cognate promoter in the pColV-K30 plasmid. We show in this article that such an unusually long operator results from the specific addition of degenerate repeats 5'-NAT(A/T)AT-3' and not from a fortuitous occupation of the DNA adjacent to the primary binding sites by an excess of the repressor. Furthermore, the protection pattern revealed by DNase I and hydroxyl radical footprinting reflected a side-by-side oligomerization of the protein along an extended DNA stretch. This type of DNA-protein interactions is more like those observed in some eukaryotic factors and nucleoid-associated proteins than typical of specific prokaryotic regulators.

5' Untranslated Regions↗
PubMed2000

Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption.

Metallothioneins (MTs) are small, cysteine-rich proteins with a strong metal-binding capacity that are ubiquitous in the animal kingdom. Recombinant expression of MT fused to outer-membrane components of gram-negative bacteria may provide new methods to treat heavy-metal pollution in industrial sewage. In this work, we have engineered Pseudomonas putida, a per se highly robust microorganism able to grow in highly contaminated habitats in order to further increase its metal-chelating ability. We report the expression of a hybrid protein between mouse MT and the beta domain of the IgA protease of Neisseria in the outer membrane of Pseudomonas cells. The metal-binding capacity of such cells was increased three-fold. The autotranslocating capacity of the beta domain of the IgA protease of Neisseria, as well as the correct anchoring of the transported protein into the outer membrane, have been demonstrated for the first time in a member of the Pseudomonas genus.

Adsorption↗
PubMed2000

Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil.

Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions. DNA sequences encoding MT were fused to the autotransporter beta-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane. The translocation, surface display, and functionality of the chimeric MTbeta protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R. eutropha. The resulting bacterial strain, named R. eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd2+-polluted soil with R. eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana).

Animals↗
PubMed2000

The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida.

In the presence of toluene and other structural analogues, the enhancer binding protein XylR activates the sigma54 promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Introduction of amino acid changes Val-219Asp and Ala-220Pro, which enter a proline kink at the interdomain region (B linker) between the A (signal reception) module and the central portion of XylR, originated a protein with unforeseen properties. These included a minor ability to activate Pu in the absence of aromatic effectors, a much higher responsiveness to m-xylene and a significant response to a large collection of aromatic inducers. Such changes could not be attributed to variations in XylR expression levels or to the fortuitous creation of a novel promoter, but to a genuine change in the properties of the activator. Structural predictions suggested that the mutation entirely disrupted an otherwise probable coiled-coil structure. A second directed mutant within the same region consisting of a major replacement of amino acids A220-N221 by the peptide HHHR produced an even more exacerbated phenotype. These data support a model in which the linker B region influences the effector profile by modifying at a distance the operative shape of the effector pocket and fixing the protein in an intermediate step of the activation process.

Alleles↗