Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein.

Which approach is more effective in the selection of plants with antimicrobial activity?

The development of the present study was based on selections using random, direct ethnopharmacological, and indirect ethnopharmacological approaches, aiming to evaluate which method is the best for bioprospecting new antimicrobial plant drugs. A crude extract of 53 species of herbaceous plants collected in the semiarid region of Northeast Brazil was tested against 11 microorganisms. Well-agar diffusion and minimum inhibitory concentration (MIC) techniques were used. Ten extracts from direct, six from random, and three from indirect ethnopharmacological selections exhibited activities that ranged from weak to very active against the organisms tested. The strain most susceptible to the evaluated extracts was Staphylococcus aureus. The MIC analysis revealed the best result for the direct ethnopharmacological approach, considering that some species yielded extracts classified as active or moderately active (MICs between 250 and 1000 µg/mL). Furthermore, one species from this approach inhibited the growth of the three Candida strains. Thus, it was concluded that the direct ethnopharmacological approach is the most effective when selecting species for bioprospecting new plant drugs with antimicrobial activities.

Initial crystallographic studies of a small heat-shock protein from Xylella fastidiosa.

The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9 kDa (HSP17.9). HSP17.9 was found as one of the proteins that are induced during X. fastidiosa proliferation and infection in citrus culture. Recombinant HSP17.9 was crystallized and surface atomic force microscopy experiments were conducted with the aim of better characterizing the HSP17.9 crystals. X-ray diffraction data were collected at 2.7 Šresolution. The crystal belonged to space group P4(3)22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 Å, and is the first small heat-shock protein to crystallize in this space group.

Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms.

The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, ß = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, ß = 94.76°.

Antimicrobial activity and bioautographic study of antistaphylococcal components from Caesalpinia pyramidalis Tull

The antimicrobial activity of dry methanol and ethyl acetate extracts for the leaves, bark of the stem, peel of the root, flower, fruit and seed of Caesalpinia pyramidalis Tull. (catingueira) was performed against seventeen isolates of Staphylococcus aureus MRSA multiresistant strains, which included two isolates of S. aureus MSSA and two ATCC strains. The antimicrobial activity was tested by the agar diffusion method and the Minimum Inhibitory Concentration (MIC) was determined. The dry methanol extract of the root showed good antimicrobial activity with a MIC of less than 0.5 mg.mL-1. The dry ethyl acetate extracts exhibited lower antimicrobial activity, which might be explained by solubility problems and less diffusion in the agar medium. Results of the bioautographies also confirmed inhibition halos corresponding to the active substances present in the leaves, as well as in the flower of C. pyramidalis. The phytochemical study of the leaves, bark of the stem, peel of the root, flower and fruit of extracts from C. pyramidalis confirmed the presence of a number of known antimicrobial agents including ursolic acid, quercetin, catechin, ellagic acid, sitosterol, flavonoids, proanthocyanidins and gallic acid.

A determinação da atividade antimicrobiana dos extratos metanólicos e em acetato de etila da folha, casca do caule, casca da raiz, flor, fruto e semente de Caesalpinia pyramidalis Tull. foi realizada frente a dezessete isolados de Staphylococcus aureus MRSA multirresistentes, dois isolados de S. aureus MSSA e duas cepas padrão, pelas técnicas de poço/difusão em ágar e determinação das CMI pelo método de diluição em agar/multiinoculador de Stears. O extrato metanólico de casca da raiz indicou uma boa atividade, com CMI inferior a 0.5 mg.mL-1. Os extratos secos por extração em acetato de etila apresentaram menor atividade que se poderia explicar por problemas de solubilidade e menor difusão no meio de cultura em ágar. Resultados das bioautografias confirmaram zonas de inibição correspondente às substâncias ativas presente na folha, como também na flor da C. pyramidalis. No estudo fitoquímico das folhas, casca da caule, casca da raiz, flor e fruto dos extratos de C. pyramidalis evidenciou-se a presença de vários constituintes com reconhecida atividade antimicrobiana, entre estes o ácido ursólico, quercetina, catequina, ácido elágico, sitosterol, flavonóides, proantocianidinas e ácido gálico. Entre todos os metabólitos citados, somente o ultimo não observamos, por CCD, na casca da raiz de C. pyramidalis.