Amalia Muñoz

Regulatory exaptation of the catabolite repression protein (Crp)-cAMP system in Pseudomonas putida.

The genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaA(P. putida) gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaA(P. putida) was poorly translated or rendered non-functional in the heterologous host. Yet, generation of cAMP by CyaA(P. putida) could be verified by expressing the cyaA(P. putida) gene in a hypersensitive E. coli strain. On the other hand, the crp(P. putida) gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress-endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp-cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co-opted for a new use.

Structure of the (113)Cd(3)beta domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms

Abstract. The three-dimensional structures of the isolated Cd3β domains from Homarusamericanus metallothionein have been determined by NMR methods in order to establish a set of β-domain structures for comparative analysis. First, it was determined that the Cd-cysteine connectivities forming the Cd3S9 metal center were identical to those observed for the βN domain in the native holoprotein. Time- and temperature-dependence studies of the 113Cd and 1H 1D-NMR spectra indicated that the βN domain undergoes slow conformational changes before reaching an equilibrium structure. In addition to structural information provided by the metal-to-cysteine connectivities, Φ, χ1 and χ2 angle constraints, three HN...S hydrogen bond interactions were also determined from a long-range optimized 1HN-113Cd HMQC experiment. A simulated annealing protocol was applied to the distance and angle constraints obtained from the 2D-NMR experiments to calculate the three-dimensional structure of the synthetic Cd3βN domain of lobster metallothionein. Structure-reactivity relationships are proposed for the reactions of Cd3β domains with 5,5′-dithiobis(2-nitrobenzoate), based on comparisons of surface exposure of sulfur atoms of the lobster and rabbit Cd3β domain structures. Finally, the surface exposure of the β domains of lobster is compared with β domains from mammalian metallothioneins. Electronic supplementary material to this paper, comprising four tables and three figures, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0345-3.

Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli.

The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic α‐helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope‐tagged version of the 23 kDa C‐terminal secretion signal of haemolysin (EHlyA). The zipper‐containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross‐linking experiments and coexpression with a secretion‐deficient derivative of ZEHlyA indicated that leucine zipper‐dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH‐domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH‐EHlyA and VHH‐ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH‐ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10‐fold increase in their avidity. These data reveal a non‐anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity.

Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

Abstract The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn7MT and Cd7MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn7MT and pyridylazoresorcinol for Cd7MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn7MT concentrations. The complex NEM concentration dependence of each phase, kf, obs=kf1+kf2 [NEM] and ks, obs=ks1+ks2 [NEM],demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested. The reactions of Cd7MT are less complete than those of Zn7MT and occur more slowly. 111Cd-NMR studies of the partially alkylated 111Cd7MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions, and rules out the possibility of cooperative reactions at either cluster.

Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting

The integration host factor (IHF) of Pseudomonas putida connects cell growth to transcriptional activity of distinct promoters. The IHF site of the σ54 promoter Pu of the TOL (m‐xylene biodegradation) plasmid pWW0 of P. putida has been examined to define experimentally a relationship between occupation of the promoter by this factor, the biological activity of the protein and the tolerance of the target site to single‐base changes through the bound DNA core sequence. The use of an in vivo high‐intensity UV imprinting procedure to examine such an occupation of Pu by IHF allowed inspection of the interplay between the factor and cognate site variants under the physiologically relevant conditions of monocopy gene dosage. The resulting data were merged in a structural model for establishing key features of the IHF–DNA interaction. A functional consensus for first‐order IHF binding was instrumental for a genome‐wide survey of sequences with potential regulatory value. This search revealed that very few, if any, of the maximum 330 sites within intergenic regions were placed in locations controlling expression of central metabolic genes. It thus seems that the IHF regulon of P. putida has a degree of functional specialization that is not evenly distributed through all gene categories.

High-temperature behavior and polymorphism in novel members of the perovskite family Pb2LnSbO6 (Ln=Ho, Er, Yb, Lu).

The synthesis, crystal structure, and dielectric properties of four novel members of the family of double perovskites Pb(2)LnSbO(6) are described. The room-temperature crystal structures were refined from neutron powder diffraction (NPD) data in the monoclinic C2/c (No. 15) space group. They contain a completely ordered array of alternating LnO(6) and SbO(6) octahedra sharing corners, tilted in antiphase along the three pseudocubic axes, with a a(-)b(-)b(-) tilting scheme, which is very unusual in the crystallochemistry of perovskites. The lead atoms occupy highly asymmetric voids with 8-fold coordination due to the stereoactivity of the Pb(2+) electron lone-pair. Several trends are observed for the entire family of compounds upon heating. The Ln = Lu, Yb, and Er oxides display three successive phase transitions in a narrow temperature range, as shown by differential scanning calorimetry (DSC) data, while the Ln = Ho shows only two transitions. Different crystal structure evolutions have been found from temperature-dependent NPD and DSC, following the space-group sequence C2/c → P2(1)/n → R ̅3 → Fm ̅3m for Ln = Lu and Yb, the sequence C2/c → unknown → P2(1)/n → Fm ̅3m for Ln = Er, and C2/c → P2(1)/n → Fm ̅3m for Ln = Ho. The Ln/Sb long-range ordering is preserved across the consecutive phase transitions. Dielectric permittivity measurements indicate the presence of a paraelectric/antiferroelectric transition (associated with the last structural transition), as suggested by the negative Curie temperature from the Curie-Weiss fit of the reciprocal permittivity.

Extreme DNA Bending: Molecular Basis of the Regulatory Breadth of IHF

The Integration host factor (IHF) is a heterodimeric, sequence-specific DNA-binding and DNA-bending protein found in many types of eubacteria. The sole function of IHF is to bring about a sharp curvature in the target DNA (up to ≥ 160°). Such a drastic change in DNA shape has been evolutionarily recruited for controlling a large number of functions that depend on the architecture of given genomic sites. These include the organization of the bacterial nucleoid and the transcriptional control of distinct promoters. The growing availability of bacterial genomes allows a comparative approach to survey the regulatory breadth of IHF in a wider context. In this Chapter, we use the sequence of the IHF protein of the soil bacterium Pseudomonas putida as a starting point to examine in detail the basis of the recognition of DNA sequences by this nucleoid-associated protein, in particular the correlation between sequence conservation and DNA interaction for each of the IHF chains. This is greatly facilitated by comparing the protein sequence and the DNA binding specificity of IHF with those of similar proteins HU and the transcription factor 1 (TF1) from bacteriophage SPO1 of Bacillus subtilis. Mapping of the fully conserved amino acids and the protein-specific sites for each chain of the corresponding tridimensional structures finely correlates with those sites involved in DNA interactions and maintaining the protein dimer structure. The sequence conservation profile of the DNA-binding regions of these proteins shows that chain B of IHF is more closely related to HU/TF1 than to chain A of IHF, suggesting a separate evolutionary origin. Furthermore, some features of the DNA recognition mechanism seem to be exclusive to IHF and cannot be fulfilled by HU or TF1 proteins. HU and TF1 can be embraced by DNA as IHF can by the action of residues conserved in the three proteins (thereby explaining why HU/TF1 and IHF can be partially replaced by each other). In contrast, only the interactions mediated by tree-determinants (i.e. those residues that are specific for each chain of IHF) can afford a high DNA recognition specificity. These analyses highlight the importance of DNA binding versus DNA bending specificities for expansion of the regulatory space of such nucleoid-associated proteins.

Exchange Reactions Between Albumin-Gold(I)-Triethylphosphine and Me(3)PAuCl or iPr(3)PAuCl(3) Studied by P NMR Spectroscopy.

The AIbSAuPEt3 complex was prepared in vitro by reacting albumin (AIbSH) with aumnofin (Et3PAuSATg). The Et3PAu entity was found to be labile to exchange in the presence f Me3PAuCI and iPraPAuCI. 31p NMR spectroscopy was used to follow these exchange reactions. Either Me3PAu or iPr3PAu + replaces Et3PAu from AIbSAuPEt3 complex. Et3PAu and R3PAu+ (R Me, iPr) were both bound to the weak binding sites (principally histidine) f the protein. Since the iPr3P ligand is bulkier than MeaPAu (reflected by its bigger Tollman cone angle) it is surprising that it replaces Et3PAu + almost equally as well as Me3PAu+.

The reaction of EDTA with metallothionein and its separate clusters: complex kinetic behavior with novel protein concentration dependence.

Mammalian metallothionein (MT) is Involved in the detoxiflcation of Cd(ll) ions. It binds 7 Cd(ll) in two Cd-thiolate clusters (Cd4S11 and C3S9) which are located in two protein domains. 111Cd and 2-D NMR studies show that in lobster, two Cd3S9 domains exist. Despite homologies in the location of cysteines between the corresponding domains of the lobster and mammalian MTs (8 of the 9 cysteines in each domain), the sequence specific Cd-thiolate connections differ in both domains of the lobster protein. To see if these differences alter the chemistry of the proteins, two reactions related to the function of the protein were compared.