Cláudia S. Oliveira

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO2)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

Thermostability of cardosin A from Cynara cardunculus L.

The structural stability of cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculusL., has been investigated by high-sensitivity differential scanning calorimetry, intrinsic fluorescence and circular dichroism spectroscopy, and enzymatic activity assays. Even though the thermal denaturation of cardosin A is partially irreversible, valid thermodynamic data can be obtained within a wide pH region. Also, although cardosin A is a heterodimeric enzyme its thermal denaturation occurs without simultaneous dissociation to unfolded monomers. Moreover, in the 3–7 pH region the excess heat capacity can be deconvoluted into two components corresponding to two elementary two-state transitions, suggesting that the two polypeptide chains of cardosin A unfold independently. Detailed thermodynamic and structural investigations of cardosin A at pH = 5.0, at which value the enzyme demonstrates maximum stability and enzymatic activity, revealed that after thermal denaturation the polypeptide chains of this protein retain most of their secondary structure motifs and are not completely hydrated.

Spatial and temporal analysis of estuarine bacterioneuston and bacterioplankton using culture-dependent and culture-independent methodologies

Bacterioneuston may play a key role in water–air exchange of gases and in processing organic matter and pollutants that accumulate at the sea-surface microlayer (SML). However, the phylogenetic diversity of bacterioneuston has been poorly characterized. We analyzed 24 samples each from the SML and underlying water (UW) at three sites in the Ria de Aveiro estuary, Portugal. Cultivation and culture-independent techniques were used to compare bacterioneuston and bacterioplankton. Culturable heterotrophic bacteria were enriched in the SML. The culturable community was dominated by Psychrobacter and Acinetobacter. The presence of high numbers of Psychrobacter was a notable result. Differences were confined to a few genera overrepresented in UW samples (Kocuria, Agrococcus and Vibrio). 16S rDNA DGGE profiles were highly stable in terms of number and position of bands between sampling sites and dates but cluster analysis revealed a slight tendency for grouping according to sampled layer. SML-specific DGGE bands affiliated with Actinobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes. Low similarity between nucleotide sequences of DGGE-bands and previously reported sequences suggest the occurrence of SML-specific populations. Enrichment of SML for Pseudomonas and Aeromonas was questioned and the diversity of both communities was analyzed. Consistent differences between SML and UW aeromonads communities were not identified. In terms of Pseudomonas, a culturable operational taxonomic unit was consistently overrepresented within SML samples. Taken together, our results indicate that the similarity between SML and UW communities depends on spatial and temporal factors.

New molecular variants of epsilon and beta IncP-1 plasmids are present in estuarine waters

In this work the presence of broad-host-plasmids in an estuary in Portugal has been investigated. Pseudomonas putida KT2442 was used as model recipient bacteria in biparental matings with tetracycline and mercury to select for resistance phenotypes. As a result, 7 transconjugants were shown to carry broad-host-plasmids from the IncP-1 group, as seen by PCR amplification of the trfA gene. Sequence analysis confirmed the isolation of 4 plasmids from β-1 subgroup and 3 assigned to the recently described ε subgroup. To our knowledge this is the first report concerning the detection and isolation of IncP-1β and ε plasmids in estuarine waters. Moreover it is shown that, even though the retrieved plasmids are phylogenetically close to previously characterized plasmids, such as pB10 and pKJK5, respectively, they constitute new molecular variants.

Co-selection of antibiotic and metal(loid) resistance in gram-negative epiphytic bacteria from contaminated salt marshes.

The goal of this study was to investigate co-selection of antibiotic resistance in gram-negative epiphytic bacteria. Halimione portulacoides samples were collected from metal(loid)-contaminated and non-contaminated salt marshes. Bacterial isolates (n=137) affiliated with Vibrio, Pseudomonas, Shewanella, Comamonas, Aeromonas and with Enterobacteriaceae. Vibrio isolates were more frequent in control site while Pseudomonas was common in contaminated sites. Metal(loid) and antibiotic resistance phenotypes varied significantly according to site contamination, and multiresistance was more frequent in contaminated sites. However, differences among sites were not observed in terms of prevalence or diversity of acquired antibiotic resistance genes, integrons and plasmids. Gene merA, encoding mercury resistance, was only detected in isolates from contaminated sites, most of which were multiresistant to antibiotics. Results indicate that metal(loid) contamination selects for antibiotic resistance in plant surfaces. In salt marshes, antibiotic resistance may be subsequently transferred to other environmental compartments, such as estuarine water or animals, with potential human health risks.

Acetonitrile-induced unfolding of porcine pepsin A: A proposal for a critical role of hydration structures in conformational stability

In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.

Comparative genomics of IncP-1ε plasmids from water environments reveals diverse and unique accessory genetic elements.

The goal of this study was to determine and compare the complete genome sequences of three new broad-host-range conjugative plasmids. Plasmids pMLUA1, pMLUA3 and pMLUA4 were previously recovered from estuarine water by exogenous plasmid isolation and ranged in size from ∼55 to 59 kb. Comparative genomics showed that their backbone region was identical to the prototype pKJK5 and other IncP1-ε plasmids captured from soils. The accessory region was inserted between the tra region and parA, and presented the typical IncP-1ε ISPa17 and Tn402-like transposon modules. Nevertheless, new class 1 integrons were identified (In794, carrying aadA5 and In795, carrying qacF5-aadA5), as well as a composite transposon IS26-msr(E)-mph(E)-IS26 carrying genes that confer resistance to macrolides. A new insertion sequence, termed ISUnCu17, was also identified on pMLUA3. The architecture of the accessory regions implies the occurrence of multiple insertions and deletions. These data support the notion that IncP-1 plasmids from the ε subgroup are proficient in the capture of diverse genetic elements, including antibiotic resistance genes, and thus may contribute to the co-selection of several resistance determinants. This study constitutes the first report of completely sequenced IncP-1ε plasmids from water environments, and enhances our understanding of the geographic distribution and genetic diversity of these replicons.

Analysis of antibiotic resistance in bacteria isolated from the surface microlayer and underlying water of an estuarine environment.

We compared the prevalence of cultivable antibiotic-resistant bacteria and resistance genes in the surface microlayer (SML) and underlying waters (UW) of an estuary. Prevalence of resistant bacteria was determined in antibiotic-supplemented agar. Bacterial isolates from the UW (n=91) and SML (n=80), selected in media without antibiotic, were characterized concerning susceptibility against nine antibiotics. The presence of genes bla(TEM), bla(OXA-B), bla(SHV), bla(IMP), tet(A), tet(B), tet(E), tet(M), cat, sul1, sul2, sul3, aadA, IntI1, IntI2, and IntI3 was assessed by PCR. The variable regions of integrons were sequenced. Ampicillin- and streptomycin-resistant bacteria were significantly more prevalent in SML. Resistance levels among the bacterial collections were generally low, preventing detection of significant differences between SML and UW. The tet(E) gene was detected in two Aeromonas isolates and tet(M) was detected in a Pseudomonas isolate. Gene sul1 was amplified from three Aeromonas isolates. Prevalence of intI genes was 2.11%. Cassette arrays contained genes encoding resistance to aminoglycosides and chloramphenicol. A higher prevalence of antibiotic-resistant bacteria in the SML, although only detectable when bacteria were selected in antibiotic-supplemented agar, suggests that SML conditions select for antibiotic resistance. Results also showed that antibiotic resistance was uncommon among estuarine bacteria and the resistance mechanisms are probably predominantly intrinsic.

Cardosin A as a model aspartic proteinase for the study of organic solvent effects. An overview on catalytic and structural aspects

Water has proven to be one of the several limitations for broadening the scope of applications of enzymes in biocatalysis, especially when the reactants involved are poorly water-soluble. The introduction of an organic solvent into the reaction system has numerous advantages. These include a direct action of the solvents on the reactants (improving their solubility) and on the reaction products (improving or diminishing their solubility), thereby increasing the productivity of the reaction system. Nevertheless, it has been documented that the introduction of an organic solvent into the reaction mixture may induce alterations on enzyme activity, enzyme stability and thermostability, and enzyme specificity thereby imprinting new properties to old enzymes.