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V de Lorenzo

Publications and source records attributed to V de Lorenzo.

At least 37 records · Page 2Linked to original sources
PubMed2000

Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system.

A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants of Escherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

Bacterial Proteins↗
PubMed2000

Functional domains of the TOL plasmid transcription factor XylS.

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

Amino Acid Sequence↗
PubMed2000

Genetic evidence of distinct physiological regulation mechanisms in the sigma(54) Pu promoter of Pseudomonas putida.

The activity of the toluene-responsive sigma(54) Pu promoter of the pWW0 TOL plasmid of Pseudomonas putida is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. Although the Pu promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of beta-galactosidase in a Pu-lacZ reporter P. putida strain. Since Pu was still down-regulated during exponential growth regardless of glucose addition, it appeared that the carbohydrate separately influenced promoter activity. This notion was supported by the growth-dependent induction pattern of Pu in a ptsN mutant of P. putida, the loss of which makes Pu no longer responsive to repression by glucose. On the other hand, overexpression of the sigma factor sigma(54), known to partially alleviate the exponential silencing of the promoter, did not affect glucose inhibition of Pu. These data indicated that exponential silencing and carbon source-dependent repression are two overlapping but genetically distinguishable mechanisms that adapt Pu to the physiological status of the cells and nutrient availability.

Culture Media↗
PubMed2000

Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.

Amino Acid Sequence↗
PubMed1999

Recruitment of RNA polymerase is a rate-limiting step for the activation of the sigma(54) promoter Pu of Pseudomonas putida.

The activity of the sigma(54)-promoter Pu of Pseudomonas putida was examined in vitro with a DNA template lacking upstream activating sequences, such that RNA polymerase can be activated by the enhancer-binding protein XylR only from solution. Although the transcription activation pathway in this system lacked the step of integration host factor (IHF)-mediated looping of the XylR.DNA complex toward the prebound RNA polymerase, IHF still stimulated promoter activity. The positive effect of IHF became evident not only with XylR from solution, but also with other sigma(54)-dependent activators such as NtrC and NifA. Furthermore, an equivalent outcome was shown for the nonspecific DNA-binding protein HU. This stimulation of transcription in the absence of the enhancer was traced to the recruitment of RNA polymerase (i.e. increased efficiency of formation of closed complexes) brought about by IHF or HU binding. Thus, under limiting concentrations of the polymerase, the factor-mediated binding of the enzyme to Pu seems to enter a kinetic checkpoint in the system that prevents the XylR-mediated formation of an open complex.

Bacterial Proteins↗
PubMed1999

The IIANtr (PtsN) protein of Pseudomonas putida mediates the C source inhibition of the sigma54-dependent Pu promoter of the TOL plasmid.

The gene cluster adjacent to the sequence of rpoN (encoding sigma factor sigma54) of Pseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of the upper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsN and ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss of ptsN, which encodes the IIANtr protein, made Pu unresponsive to repression by glucose. The ptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directed ptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some sigma54 promoters in which IIANtr is a key protein intermediate.

Bacterial Proteins↗
PubMed1999

Involvement of the FtsH (HflB) protease in the activity of sigma 54 promoters.

The effect of FtsH, an essential inner membrane-bound protease, in the regulation of the sigma 54-dependent Pu promoter has been examined in vivo. Escherichia coli cells lacking FtsH failed to activate a Pu-lacZ fusion in response to the cognate enhancer-binding protein XylR. However, the intracellular concentrations of XylR and sigma 54, as well as their apparent physical integrity were the same regardless of the presence or absence of the protease. The loss of Pu activity in FtsH-minus cells was not due to the imbalance between sigma factors caused by the lack of the protease. ftsH mutants could not grow in media with glutamine as the only nitrogen source and failed also to induce the sigma 54 promoters PnifH by NifA and PpspA by PspF. These lesions were fully complemented by a ftsH+ plasmid. Therefore, part of the pleiotropic phenotype of FtsH-less cells corresponded to the lack of sigma 54 activity. Overproduction of sigma 54, however, restored both transcriptional activity of Pu and growth in glutamine of a ftsH strain. These observations suggested that the activity of sigma 54 is checked in vivo by an interplay of factors that ultimately determine the performance of cognate promoters under given physiological conditions.

ATP-Dependent Proteases↗
PubMed1999

Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain.

The mechanism of protein secretion mediated by the beta-domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram-negative bacteria called autotransporters, has been examined using a single-chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the beta-module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N-terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S-S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N-passenger domain can be translocated through the beta-domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re-evaluation of the prevailing model for secretion of the N-domain of autotransporters.

Animals↗
PubMed1999

Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous two-liquid-phase applications.

Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.

Bacteriological Techniques↗
PubMed1999

Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides.

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

Amino Acid Sequence↗
PubMed1998

Binding of the fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence.

The mode of DNA binding of the Fur (ferric uptake regulator) repressor which controls transcription of iron-responsive genes in Escherichia coli, has been re-examined. Using as a reference the known sites at the promoter of the aerobactin operon of Escherichia coli, we have compared in detail the patterns of interaction between the purified Fur protein and natural or synthetic DNA targets. DNase I and hydroxyl radical footprinting, as well as missing-T assays, consistently revealed that functional Fur sites are composed of a minimum of three repeats of the hexameric motif GATAAT rather than by a palindromic 19 bp target sequence. Extended binding sites, constructed by stepwise addition of one or two direct repeats of the same sequence, were occupied co-operatively by Fur with the same pattern of interactions as those observed with the core of three repeats. This indicated that functional sites with a range of affinities can be formed by the addition of discrete GATAAT extensions to a minimal recognition sequence. The fashion in which Fur binds its target, virtually unknown in prokaryotic transcriptional regulators, accounts for the observed helical wrapping of the protein around the DNA helix.

Bacterial Proteins↗
PubMed1998

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.

Bacterial Proteins↗
PubMed1998

Expression systems and physiological control of promoter activity in bacteria.

Promoter activity in vivo is not just dependent on the performance of the regulator/promoter pair which may predominantly control transcription initiation in response to a given signal, it also relies on overimposed mechanisms that connect the activity of individual promoters to the metabolic and energetic status of the bacterial cells. Such mechanisms - which frequently become limiting for biotechnological applications involving regulated promoters - include classic (i.e. cAMP/CRP-mediated) or alternative catabolite control checks, recruitment of protein intermediates of the phosphotransferase sugar transport system, coregulation through protein-induced DNA bending and the interplay of sigma factors during various growth stages.

Cyclic AMP Receptor Protein↗
PubMed1998

Activation of the toluene-responsive regulator XylR causes a transcriptional switch between sigma54 and sigma70 promoters at the divergent Pr/Ps region of the TOL plasmid.

The mechanism by which XylR, the toluene-responsive activator of the sigma54-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own sigma70 promoter Prhas been examined. An in vitro transcription system was developed in order to reproduce the repression of Probserved in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two sigma70-RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent sigma54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N-terminal domain (XylRdeltaA). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent sigma54 promoter Ps during Pr downregulation by XylRdeltaA. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and Ps. Such a switch is apparently caused by the ATP-dependent multimerization and strong DNA binding of the protein required for activation of the sigma54 promoter. This device could reset the level of XylR expression during activation of the sigma54 Pu and Ps promoters of the TOL plasmid.

Adenosine Triphosphate↗
PubMed1998

Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway.

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of a P. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.

Biotransformation↗
PubMed1998

Coordinated repression in vitro of the divergent fepA-fes promoters of Escherichia coli by the iron uptake regulation (Fur) protein.

The mechanism involved in transcriptional repression of the fepA-fes divergent promoters of Escherichia coli by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped -35/-10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers was fully prevented by Fur-Mn2+ bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the fes and fepA promoters could be displaced by Fur-Mn2+, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the fes promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the fes and fepA promoters and their coordinate regulation regardless of their different transcriptional activities. Comparisons with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.

Bacterial Outer Membrane Proteins↗