Genomic prediction with allele dosage information in highly polyploid species.

KEY MESSAGE: Including allele, dosage can improve genomic selection in highly polyploid species under higher frequency of different heterozygous genotypic classes and high dominance degree levels. Several studies have shown how to leverage allele dosage information to improve the accuracy of genomic selection models in autotetraploid. In this study, we expanded the methodology used for genomic selection in autotetraploid to higher (and mixed) ploidy levels. We adapted the models to build covariance matrices of both additive and digenic dominance effects that are subsequently used in genomic selection models. We applied these models using estimates of ploidy and allele dosage to sugarcane and sweet potato datasets and validated our results by also applying the models in simulated data. For the simulated datasets, including allele dosage information led up to 140% higher mean predictive abilities in comparison to using diploidized markers. Including dominance effects were highly advantageous when using diploidized markers, leading to mean predictive abilities which were up to 115% higher in comparison to only including additive effects. When the frequency of heterozygous genotypes in the population was low, such as in the sugarcane and sweet potato datasets, there was little advantage in including allele dosage information in the models. Overall, we show that including allele dosage can improve genomic selection in highly polyploid species under higher frequency of different heterozygous genotypic classes and high dominance degree levels.

Coexpression and Transcriptome analyses identify active Apomixis-related genes in Paspalum notatum leaves.

BACKGROUND: Paspalum notatum exhibits both sexual and apomictic cytotypes and, thus, is considered a good model for studies of apomixis because it facilitates comparative approaches. In this work, transcriptome sequencing was used to compare contrasting P. notatum cytotypes to identify differential expression patterns and candidate genes involved in the regulation of expression of this trait. RESULTS: We built a comprehensive transcriptome using leaf and inflorescence from apomictic tetraploids and sexual diploids/tetraploids and a coexpression network based on pairwise correlations between transcript expression profiles. We identified genes exclusively expressed in each cytotype and genes differentially expressed between pairs of cytotypes. Gene Ontology enrichment analyses were performed to better interpret the data. We de novo assembled 114,306 reference transcripts. In total, 536 candidate genes possibly associated with apomixis were detected through statistical analyses of the differential expression data, and several interacting genes potentially linked to the apomixis-controlling region, genes that have already been reported in the literature, and their neighbors were transcriptionally related in the coexpression network. CONCLUSIONS: Apomixis is a highly desirable trait in modern agriculture due to the maintenance of the characteristics of the mother plant in the progeny. The reference transcriptome, candidate genes and their coexpression network identified in this work represent rich resources for future grass breeding programs.

"Integrative genomic analysis of the bioprospection of regulators and accessory enzymes associated with cellulose degradation in a filamentous fungus (Trichoderma harzianum)".

BACKGROUND: Unveiling fungal genome structure and function reveals the potential biotechnological use of fungi. Trichoderma harzianum is a powerful CAZyme-producing fungus. We studied the genomic regions in T. harzianum IOC3844 containing CAZyme genes, transcription factors and transporters. RESULTS: We used bioinformatics tools to mine the T. harzianum genome for potential genomics, transcriptomics, and exoproteomics data and coexpression networks. The DNA was sequenced by PacBio SMRT technology for multiomics data analysis and integration. In total, 1676 genes were annotated in the genomic regions analyzed; 222 were identified as CAZymes in T. harzianum IOC3844. When comparing transcriptome data under cellulose or glucose conditions, 114 genes were differentially expressed in cellulose, with 51 being CAZymes. CLR2, a transcription factor physically and phylogenetically conserved in Trichoderma spp., was differentially expressed under cellulose conditions. The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family. CONCLUSIONS: Our results provide new insights into the relationship between genomic organization and hydrolytic enzyme expression and regulation in T. harzianum IOC3844. Our results can improve plant biomass degradation, which is fundamental for developing more efficient strains and/or enzymatic cocktails to produce hydrolytic enzymes.

Microsatellites for the Neotropical Ant, Odontomachus chelifer (Hymenoptera: Formicidae).

Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer's genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna.

InP Nanowire Biosensor with Tailored Biofunctionalization: Ultrasensitive and Highly Selective Disease Biomarker Detection.

Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities ∼1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of ∼6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices.

Production of a recombinant swollenin from Trichoderma harzianum in Escherichia coli and its potential synergistic role in biomass degradation.

BACKGROUND: Fungal swollenins (SWOs) constitute a class of accessory proteins that are homologous to canonical plant expansins. Expansins and expansin-related proteins are well known for acting in the deagglomeration of cellulose structure by loosening macrofibrils. Consequently, SWOs can increase the accessibility and efficiency of the other enzymes involved in the saccharification of cellulosic substrates. Thus, SWOs are promising targets for improving the hydrolysis of plant biomass and for use as an additive to enhance the efficiency of an enzyme cocktail designed for the production of biofuels. RESULTS: Here, we report the initial characterization of an SWO from Trichoderma harzianum (ThSwo) that was successfully produced using Escherichia coli as a host. Initially, transcriptome and secretome data were used to compare swo gene expression and the amount of secreted ThSwo. The results from structural modeling and phylogenetic analysis of the ThSwo protein showed that ThSwo does preserve some structural features of the plant expansins and family-45 glycosyl hydrolase enzymes, but it evolutionarily diverges from both of these protein classes. Recombinant ThSwo was purified at a high yield and with high purity and showed secondary folding similar to that of a native fungal SWO. Bioactivity assays revealed that the purified recombinant ThSwo created a rough and amorphous surface on Avicel and displayed a high synergistic effect with a commercial xylanase from T. viride, enhancing its hydrolytic performance up to 147 ± 7%. CONCLUSIONS: Many aspects of the structure and mechanism of action of fungal SWOs remain unknown. In the present study, we produced a recombinant, active SWO from T. harzianum using a prokaryotic host and confirmed its potential synergistic role in biomass degradation. Our work paves the way for further studies evaluating the structure and function of this protein, especially regarding its use in biotechnology.

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development.

Species distribution and introgressive hybridization of two Avicennia species from the Western Hemisphere unveiled by phylogeographic patterns.

BACKGROUND: Mangrove plants grow in the intertidal zone in tropical and subtropical regions worldwide. The global latitudinal distribution of the mangrove is mainly influenced by climatic and oceanographic features. Because of current climate changes, poleward range expansions have been reported for the major biogeographic regions of mangrove forests in the Western and Eastern Hemispheres. There is evidence that mangrove forests also responded similarly after the last glaciation by expanding their ranges. In this context, the use of genetic tools is an informative approach for understanding how historical processes and factors impact the distribution of mangrove species. We investigated the phylogeographic patterns of two Avicennia species, A. germinans and A. schaueriana, from the Western Hemisphere using nuclear and chloroplast DNA markers. RESULTS: Our results indicate that, although Avicennia bicolor, A. germinans and A. schaueriana are independent lineages, hybridization between A. schaueriana and A. germinans is a relevant evolutionary process. Our findings also reinforce the role of long-distance dispersal in widespread mangrove species such as A. germinans, for which we observed signs of transatlantic dispersal, a process that has, most likely, contributed to the breadth of the distribution of A. germinans. However, along the southern coast of South America, A. schaueriana is the only representative of the genus. The distribution patterns of A. germinans and A. schaueriana are explained by their different responses to past climate changes and by the unequal historical effectiveness of relative gene flow by propagules and pollen. CONCLUSIONS: We observed that A. bicolor, A. germinans and A. schaueriana are three evolutionary lineages that present historical and ongoing hybridization on the American continent. We also inferred a new evidence of transatlantic dispersal for A. germinans, which may have contributed to its widespread distribution. Despite the generally wider distribution of A. germinans, only A. schaueriana is found in southern South America, which may be explained by the different demographic histories of these two species and the larger proportion of gene flow produced by propagules rather than pollen in A. schaueriana. These results highlight that these species responded in different ways to past events, indicating that such differences may also occur in the currently changing world.

Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/ß fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress.

Molecular genetic variability of commercial and wild accessions of passion fruit (Passiflora spp.) targeting ex situ conservation and breeding.

Passiflora species are distributed throughout Latin America, and Brazil and Colombia serve as the centers of diversity for this genus. We performed cross-species amplification to evaluate 109 microsatellite loci in 14 Passiflora species and estimated the diversity and genetic structure of Passiflora cincinnata, Passiflora setaceae and Passiflora edulis. A total of 127 accessions, including 85 accessions of P. edulis, a commercial species, and 42 accessions of 13 wild species, were examined. The cross-species amplification was effective for obtaining microsatellite loci (average cross-amplification of 70%). The average number of alleles per locus (five) was relatively low, and the average diversity ranged from 0.52 in P. cincinnata to 0.32 in P. setacea. The Bayesian analyses indicated that the P. cincinnata and P. setacea accessions were distributed into two groups, and the P. edulis accessions were distributed into five groups. Private alleles were identified, and suggestions for core collections are presented. Further collections are necessary, and the information generated may be useful for breeding and conservation.

Genetic breeding and diversity of the genus Passiflora: progress and perspectives in molecular and genetic studies.

Despite the ecological and economic importance of passion fruit (Passiflora spp.), molecular markers have only recently been utilized in genetic studies of this genus. In addition, both basic genetic researches related to population studies and pre-breeding programs of passion fruit remain scarce for most Passiflora species. Considering the number of Passiflora species and the increasing use of these species as a resource for ornamental, medicinal, and food purposes, the aims of this review are the following: (i) to present the current condition of the passion fruit crop; (ii) to quantify the applications and effects of using molecular markers in studies of Passiflora; (iii) to present the contributions of genetic engineering for passion fruit culture; and (iv) to discuss the progress and perspectives of this research. Thus, the present review aims to summarize and discuss the relationship between historical and current progress on the culture, breeding, and molecular genetics of passion fruit.

Population genetics and genomics of Triatoma brasiliensis (Hemiptera, Reduviidae) in an area of high pressure of domiciliary infestation in Northeastern Brazil.

Understanding the population dynamics of vectors is crucial for effective control of vector-borne diseases. In the Northeastern Brazilian semi-arid region, Triatoma brasiliensis persists as the most significant Chagas disease vector, frequently displaying recurrent domiciliary infestations. This situation raises relevant public health concerns in the municipality of Currais Novos in the state of Rio Grande do Norte. This area has experienced a high prevalence of peridomiciliary re-infestations by T. brasiliensis, coupled with elevated rates of Trypanosoma cruzi infection. Therefore, we assessed the distribution of genetic variation via mitochondrial Cytochrome b gene (MT-CYB) sequencing (n = 109) and single nucleotide polymorphisms (SNPs, n = 86) to assess the gene flow among distinct populations distributed in varied geographic spots and environments, mainly sylvatic and peridomiciliary. Insects were collected from rural communities at Currais Novos, enclosed within a 16 km radius. Sampling included 13 populations: one intradomiciliary, eight peridomiciliary, and four sylvatic. Furthermore, an external population located 220 km from Currais Novos was also included in the study. The method employed to obtain SNP information relied on ddRAD-seq genotyping-by-sequencing (GBS), enabling a genome-wide analysis to infer genetic variation. Through AMOVA analysis of MT-CYB gene variation, we identified four distinct population groups with statistical significance (FCT= 0.42; p<0.05). We identified a total of 3,013 SNPs through GBS, with 11 loci showing putative signs of being under selection. The variation based on 3,002 neutral loci evidenced low genetic structuration based on low FST values (p>0.05), indicating local panmixia. However, resampling algorithms pointed out that three samples from the external population were assigned (>98 %) in a cluster contrasting from the ones putatively under local panmixia - validating the newly applied genome-wide marker for studies on the population genetics at finer-scale resolution for T. brasiliensis. The presence of population structuring in some of the sampled points, as suggested by the mitochondrial marker, leads us to assume that infestations were probably initiated by small populations of females - demographic event poses a risk for rapid re-infestations. The local panmictic pattern revealed by the GBS marker poses a challenge for vector control measures, as re-infestation foci may be distributed over a wide geographical and ecological range. In such instances, vectors exhibit reduced susceptibility to conventional insecticide spraying operations since sylvatic populations are beyond the reach of these interventions. The pattern of infestation exhibited by T. brasiliensis necessitates integrating innovative strategies into the existing control framework, holding the potential to create a more resilient and adaptive vector control program. In our dataset, the results demonstrated that the genetic signals from both markers were complementary. Therefore, it is essential to consider the nature and inheritance pattern of each marker when inferring the pattern of re-infestations.

Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity.

Genetic variation in polyploid forage grass: assessing the molecular genetic variability in the Paspalum genus.

BACKGROUND: Paspalum (Poaceae) is an important genus of the tribe Paniceae, which includes several species of economic importance for foraging, turf and ornamental purposes, and has a complex taxonomical classification. Because of the widespread interest in several species of this genus, many accessions have been conserved in germplasm banks and distributed throughout various countries around the world, mainly for the purposes of cultivar development and cytogenetic studies. Correct identification of germplasms and quantification of their variability are necessary for the proper development of conservation and breeding programs. Evaluation of microsatellite markers in different species of Paspalum conserved in a germplasm bank allowed assessment of the genetic differences among them and assisted in their proper botanical classification. RESULTS: Seventeen new polymorphic microsatellites were developed for Paspalum atratum Swallen and Paspalum notatum Flüggé, twelve of which were transferred to 35 Paspalum species and used to evaluate their variability. Variable degrees of polymorphism were observed within the species. Based on distance-based methods and a Bayesian clustering approach, the accessions were divided into three main species groups, two of which corresponded to the previously described Plicatula and Notata Paspalum groups. In more accurate analyses of P. notatum accessions, the genetic variation that was evaluated used thirty simple sequence repeat (SSR) loci and revealed seven distinct genetic groups and a correspondence of these groups to the three botanical varieties of the species (P. notatum var. notatum, P. notatum var. saurae and P. notatum var. latiflorum). CONCLUSIONS: The molecular genetic approach employed in this study was able to distinguish many of the different taxa examined, except for species that belong to the Plicatula group, which has historically been recognized as a highly complex group. Our molecular genetic approach represents a valuable tool for species identification in the initial assessment of germplasm as well as for characterization, conservation and successful species hybridization.

A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.

A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/ß hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495µM and 0.24µM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

Are There Edge Effects on the Genetic Diversity of the Trap-Jaw Ant Odontomachus chelifer (Formicidae: Ponerinae) in a Neotropical Savanna Fragment? A First Assessment.

Habitat fragmentation is considered an important threat to biodiversity, increasing species exposure to edge effects. The Brazilian Cerrado savanna is considered a biodiversity hotspot and has been converted to small, isolated fragments due to human activities. Ant communities and colony survivorship are known to be affected by edge effects in Cerrado, but to date there is no information on the genetic diversity of ant colonies at the edge of fragmented areas. Here, we investigate if colony genetic diversity and structure of Odontomachus chelifer (Latreille) ants (Hymenoptera: Formicidae) are subject to edge effects in a Cerrado reserve in southeast Brazil. Using microsatellites, we evaluated the number of breeders (queens and males) and the genetic diversity in O. chelifer colonies located in the interior versus edge of a Cerrado fragment. All O. chelifer nests had multiple queens, which presented a low mating frequency. The number of breeders and most estimates of genetic diversity did not differ between colonies at the edge versus interior of the fragment. Genetic structure was not influenced by nest location as well. However, we detected a small and positive increase in the observed heterozygosity in colonies located at fragment edges. High heterozygosity is thought to be particularly important in fast-changing environments, such as edges, providing an advantage for genetic diversity. Further investigation is needed to assess in greater detail how habitat loss affects O. chelifer biology. Our study is a first step toward elucidating edge effects on genetic diversity of ant colonies, a topic still poorly explored in tropical environments.

A molecular linkage map for Drosophila mediopunctata confirms synteny with Drosophila melanogaster and suggests a region that controls the variation in the number of abdominal spots.

The classic approach to gene discovery relies on the construction of linkage maps. We report the first molecular-based linkage map for Drosophila mediopunctata, a neotropical species of the tripunctata group. Eight hundred F(2) individuals were genotyped at 49 microsatellite loci, resulting in a map that is ≈450 centimorgans long. Five linkage groups were detected, and the species' chromosomes were identified through cross-references to BLASTn searches and Müller elements. Strong synteny was observed when compared with the Drosophila melanogaster chromosome arms, but little conservation in the gene order was seen. The incorporation of morphological data corresponding to the number of central abdominal spots on the map was consistent with the expected location of a genomic region responsible for the phenotype on the second chromosome.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as ß-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures.

A novel linkage map of sugarcane with evidence for clustering of retrotransposon-based markers.

BACKGROUND: The development of sugarcane as a sustainable crop has unlimited applications. The crop is one of the most economically viable for renewable energy production, and CO2 balance. Linkage maps are valuable tools for understanding genetic and genomic organization, particularly in sugarcane due to its complex polyploid genome of multispecific origins. The overall objective of our study was to construct a novel sugarcane linkage map, compiling AFLP and EST-SSR markers, and to generate data on the distribution of markers anchored to sequences of scIvana_1, a complete sugarcane transposable element, and member of the Copia superfamily. RESULTS: The mapping population parents ('IAC66-6' and 'TUC71-7') contributed equally to polymorphisms, independent of marker type, and generated markers that were distributed into nearly the same number of co-segregation groups (or CGs). Bi-parentally inherited alleles provided the integration of 19 CGs. The marker number per CG ranged from two to 39. The total map length was 4,843.19 cM, with a marker density of 8.87 cM. Markers were assembled into 92 CGs that ranged in length from 1.14 to 404.72 cM, with an estimated average length of 52.64 cM. The greatest distance between two adjacent markers was 48.25 cM. The scIvana_1-based markers (56) were positioned on 21 CGs, but were not regularly distributed. Interestingly, the distance between adjacent scIvana_1-based markers was less than 5 cM, and was observed on five CGs, suggesting a clustered organization. CONCLUSIONS: Results indicated the use of a NBS-profiling technique was efficient to develop retrotransposon-based markers in sugarcane. The simultaneous maximum-likelihood estimates of linkage and linkage phase based strategies confirmed the suitability of its approach to estimate linkage, and construct the linkage map. Interestingly, using our genetic data it was possible to calculate the number of retrotransposon scIvana_1 (~60) copies in the sugarcane genome, confirming previously reported molecular results. In addition, this research possibly will have indirect implications in crop economics e.g., productivity enhancement via QTL studies, as the mapping population parents differ in response to an important fungal disease.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.