Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B₁ PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

     

Song‐based species discrimination in a rapid Neotropical radiation of grassland seedeaters

Acoustic signals among newly diverged taxa have the potential to convey species identity, information that is key to reducing hybridization. Capuchino seedeaters constitute a remarkable example of recently radiated endemic species from the grasslands of South America. They are sexually dimorphic and show striking differences in male plumage coloration and song. Contrasting with this divergence in phenotype most species show extremely low neutral genetic differentiation and lack of reciprocal monophyly, which is interpreted to be a product of recent common ancestry and hybridization. Here we use field‐based playback experiments to test for the first time if males of two species, Sporophila hypoxantha and S. palustris, discriminate between conspecific and heterospecific song. Using various measures of behavior we find that both species react more strongly to their own songs. The response to playback from another southern capuchino cannot be differentiated from that of a control song from a more distantly related Sporophila species. Additionally, we did not find evidence for reinforcement as the response of S. hypoxantha did not differ between individuals that co‐occur with S. palustris and those that do not. Our finding suggests that song, a culturally inherited trait, may help maintain reproductive isolation between species in the rapid and explosive capuchino radiation.     

Analysis from avian visual perspective reveals plumage colour differences among females of capuchino seedeaters (Sporophila)

Females of the closely related capuchino seedeaters are difficult to distinguish from one another based on human visual perception of colouration and morphology. We examined plumage colour differences among females of four species, the tawny‐bellied seedeater Sporophila hypoxantha, the dark‐throated seedeater S. ruficollis, the rufous‐rumped seedeater S. hypochroma, and the chesnut‐bellied seedeater S. cinnamomea. Reflectance values were measured on museum skins, and interspecific differences were analyzed using the Vorobyev‐Osorio avian colour discrimination model. Interspecific distances in the colour space calculated by the model were considerably higher than the threshold for colour discrimination, indicating the presence of colour differences among species that should be detectable by birds. Colour differences between S. hypoxantha and the other three species were the largest. A Discriminant Function Analysis showed that UV‐wavelength was particularly important in species separation. Our results indicate that female plumage of these four species is considerably divergent in colour; these differences being imperceptible to the human eye, thus representing previously unknown morphological evolution in these species.     

Changes in the feeding ecology of South American sea lions on the southern Brazilian coast over the last two decades of excessive fishing exploration

In the last decades, an increasing fishing effort and a decreasing trend in fish catches have been observed in southern Brazil. Considering that marine mammals and fisheries usually compete for the same resources, it is reasonable to presume that the feeding ecology of these predators is affected by the current scenario. To evaluate this hypothesis, long-term variation in the diet of the South American sea lion (Otaria flavescens) relative to fisheries exploitation was analyzed for two periods (1993–2003 versus 2004–2014). The degree of overlap between the relative biomass of the sea lions’ diet and the target species of six types of local fishery was analyzed. An increase in prey overlap between sea lions and fisheries was observed in the more recent sampling period, along with an increase in prey diversity, richness, and niche breadth of the sea lions’ diet. These results suggest that the overfishing scenario could partly explain the modified feeding ecology of the sea lions. In this context, we recommend a review and better regulation of the current fishing effort in the region, which we believe will be an important step to maintain the fish stocks and minimize the impact of fishing on marine top predators.     

Local adaptation versus historical isolation as sources of melanin‐based coloration in the white‐throated thrush Turdus assimilis

Local adaptation seems to be one of the causes of variation in melanin‐based colors in bird plumages, related mainly to the heterogeneity of the environmental conditions along the distribution of a species. Based on comparisons of genetic (mtDNA sequences), ecological (niche models), and quantitative colorimetric data, we explored variation in plumage coloration of the white‐throated thrush Turdus assimilis, a Mesoamerican species whose dorsal color varies from brown (northern and central Mexico) to dark gray (southern Mexico and Central America). Our results suggest the existence of two major patterns of coloration in this bird, which are congruent with the genetic structure, and comparisons of ecological niche models showed that population's niches were more similar than expected by chance, suggesting that color variation in plumage of T. assimilis is not consequence of local adaptation to different environmental conditions. Our results also showed that a greater geographic distance between populations is correlated with greater colorimetric differences, suggesting that color variation in T. assimilis may be consequence of historical isolation.     

Postnatal development of franciscana's (Pontoporia blainvillei) biosonar relevant structures with potential implications for function,life history,and bycatch

Franciscana dolphins (Pontoporia blainvillei) are the most endangered species of the western South Atlantic Ocean. The major cause of their vulnerability is incidental bycatch in fishery gill nets. Ontogenetic changes of biosonar relevant structures in Pontoporia were analyzed in five specimens (one female neonate, two male neonates and two male adults) using digital imaging technology (MRI, CT) and macroscopic dissections to compare structures involved in sound production and reception. These data were compared to an ontogenetic series of 69 macerated skulls of Pontoporia in order to elucidate the correlation between soft tissue structures and bones of the epicranial complex and to describe the development‐related changes in the mandible. Postnatal developmental shape changes of the posterior part of the right vestibular air sac followed bone formation and the melon with its right branch elongated, paralleling the flatter facial depression of adults. Minor postnatal developmental modifications were verified in the tympano‐periotic complex but a shape change of the mandible was visible by a ventral deviation of the posterior part of the mandible in adults. These results reveal postnatal changes in allometry and shape of biosonar relevant structures that may be one of the causes that increase bycatch of neonate and young Pontoporia individuals.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Evolutionary walk between (β/α)(8) barrels: catalytic migration from triosephosphate isomerase to thiamin phosphate synthase

The functionally versatile (β/α)(8) barrel scaffold was used to migrate triosephosphate isomerase (TPI) to thiamin phosphate synthase (TPS) activity, two enzymes that share the same fold but catalyze unrelated reactions through different mechanisms. The high sensitivity of the selection methodology was determinant to succeed in finding proteins with the desired activity. A combination of rational design and random mutagenesis was used to achieve the desired catalytic migration. One of the parallel directed evolution strategies followed resulted in TPI derivatives able to complement the thiamin phosphate auxotrophy phenotype of an Escherichia coli strain deleted of thiE, the gene that codes for TPS. Successive rounds of directed evolution resulted in better complementing TPI variants. Biochemical characterization of some of the evolved TPI clones demonstrated that the K(m) for the TPS substrates was similar to that of the native TPS; however and in agreement with the very slow complementation phenotype, the k(cat) was 4 orders of magnitude lower, indicating that substrate binding played a major role on selection. Interestingly, the crystal structure of the most proficient variant showed a slightly modified TPI active site occupied by a thiamin-phosphate-like molecule. Substitution of key residues in this region reduced TPS activity, strongly suggesting that this is also the catalytic site for the evolved TPS activity. The presence of the TPS reaction product at the active site explains the fast inactivation of the enzyme observed. In conclusion, by combining rational design, random mutagenesis and a very sensitive selection, it is possible to achieve enzymatic activity migration.     

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.