Beatriz Maestro

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae.

The eukaryotic‐type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal‐transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C‐terminal domain of StkP kinase (C‐StkP), elaborated a three‐dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C‐StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β‐lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA‐containing kinase, suggesting that non‐crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β‐lactam antibiotics.

Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors.

Snail proteins are C2H2 class zinc finger transcription factors involved in different processes during embryonic development, as well as in several adult pathologies including cancer and organ fibrosis. The expression of Snail transcription factors is tightly regulated at the transcriptional level and their activity is modulated by their subcellular localization. Given the importance of this gene family in physiology and pathology, it is essential to understand the mechanisms by which Snail proteins are imported into or exported out of the nucleus. Here we show that several importins mediate the nuclear import of the human Snail proteins and we identify a unique nuclear localization signal (NLS), recognized by all the importins, that has been conserved during the evolution of the Snail family. This NLS is characterized by the presence of basic residues at defined positions in at least three consecutive zinc fingers. Interestingly, the consensus residues for importin-binding are also involved in DNA binding, suggesting that importins could prevent non-specific binding of these transcription factors to cytoplasmic polyanions. Importantly, the identified basic residues are also conserved in other families of C2H2 transcription factors whose nuclear localization requires the zinc finger region.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442.

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein

We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37 degrees C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than E. coli such as Pseudomonas putida and Alcaligenes faecalis. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA.

Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems.

We present a novel procedure for affinity partitioning of recombinant proteins fused to the choline-binding module C-LytA in aqueous two-phase systems containing poly(ethylene glycol) (PEG). Proteins tagged with the C-LytA module and exposed to the two-phase systems are quantitatively localized in the PEG-rich phase, whereas subsequent addition of the natural ligand choline specifically shifts their localization to the PEG-poor phase by displacement of the polymer from the binding sites. The described procedure is simple, scalable and reproducible, and has been successfully applied to the purification of four diverse proteins, resulting in high yields and purity.

Inhibition of pneumococcal choline‐binding proteins and cell growth by esters of bicyclic amines

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide‐based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline‐binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline‐binding module (C‐LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C‐LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C‐LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline‐dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

Multivalent Choline Dendrimers as Potent Inhibitors of Pneumococcal Cell-Wall Hydrolysis†

Cholin bindende Proteine aus Streptococcus pneumoniae erkennen unterschiedliche Cholin-Architekturen auf bakteriellen Zellwanden. Cholinfunktionalisierte Dendrimere hemmen diese lebenswichtigen Enzyme stark, und mit einer 103- bis 104-fach hoheren Affinitat als freies Cholin: So werden Autolyse und Zelltrennung in Bakterienkulturen durch Konzentrationen im niedrigen mikromolaren Bereich inhibiert (siehe Bild).

Novel approaches to fight Streptococcus pneumoniae.

Streptococcus pneumoniae affects millions of people worldwide. It is responsible for a wide spectrum of serious illnesses such as pneumonia, meningitis and bacteraemia. The highest rate of pneumococcal disease (and the highest mortality) occurs in young children, as well as in the elderly and the immunocompromised patients. Identification of S. pneumoniae in diagnostic procedures may significantly improve thanks to the descripion of new PCR-derived techniques. Vaccination based on the polysaccharidic capsule, together with benzylpenicillin-derived drugs, constitute the current choices to tackle pneumococcal diseases. However, the wide serotype diversity of S. pneumoniae and the emergence of antibiotic-resistant strains is fostering the development of new methods to fight this microorganism. In this sense, patents documenting the use of novel antibiotics of the fluoroquinolone or tetracycline families have recently been described. Moreover, surface-associated proteins are receiving an increasingly special attention, as they are synthesized by most pneumococcal strains and play an important role in virulence. New patented protein-based vaccines take into consideration these polypeptides. In this article we present the main relevant characteristics of this pathogen and review the most recent methods that have been patented for the prevention, diagnostic and treatment of the pneumococcal diseases.

Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed beta-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 degrees C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)-->I1<=>I2. The N-->I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1<=>I2 transition is co-operative and reversible, with an associated freeenergy change (DeltaG(0)) of 30.9+/-0.8 kJ x mol(-1). The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2<=>[I2]2, DeltaG(0)=50.1+/-0.8 kJ x mol(-1)). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal beta-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.

Modulation of pPS10 host range by DnaA

Narrow‐host‐range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E. coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As the E. coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E. coli. The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E. coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establish‐ment of pPS10 in that bacteria and probably in other species.