Dynamics of the role of LacMeta laccase in the complete degradation and detoxification of malachite green.

Laccases highlight for xenobiotic bioremediation, as well as application in the fine chemical, textile, biofuel and food industries. In a previous work, we described the preliminary characterization of laccase LacMeta, a promising enzyme for the bioremediation of dyes, able to decolorization malachite green (MG), trypan blue, methylene blue. Here we demonstrate that LacMeta is indeed suitable for the complete degradation and detoxification of MG dye, not just for its discoloration, since some works show false positives due to the formation of colorless intermediates such as leucomalachite. The optimal pH and temperature parameters of LacMeta were 5.0 and 50 °C, respectively (MG as substrate). LacMeta was tolerant of up to 10 mmol L- 1 EDTA (82%) and up to 5% (V/V) acetone (91%) and methanol (71%), while SDS promoted severe inhibition. For ions, a high tolerance to cobalt, zinc, manganese, and calcium (10 mmol L- 1) was also observed (> 90%). Even under high-salinity conditions (1 mol L- 1 NaCl), the residual bleaching activity of the dye remained at 61%. Furthermore, the bleaching product of MG did not inhibit the germination of sorghum and tomato seeds and was inert to the vegetative structures of the germinated seedlings. Additionally, this treatment effectively reduced the cytotoxic effect of the dye on microorganisms (Escherichia coli and Azospirillum brasilense), which can be explained by H-NMR spectral analysis results since LacMeta completely degraded the peak signals corresponding to the aromatic rings in the dye, demonstrating extreme efficiency in the bioremediation of the xenobiotic at high concentrations (50 mg L- 1).

Broad thermal spectrum metagenomic laccase with action for dye decolorization and fentin hydroxide treatment.

Laccases are multicopper oxidases that act on various phenolic and non-phenolic compounds, enabling numerous applications including xenobiotic bioremediation, biofuel production, drug development, and cosmetic production, and they can be used as additives in the textile and food industries. This wide range of uses makes these enzymes extremely attractive for novel biotechnology applications. Here, we undertook the kinetic characterization of LacMeta, a predicted as homotrimeric (~ 107,93 kDa) small laccase, and demonstrated that this enzyme performs best at an acidic pH (pH 3-5) towards ABTS as substrate and has a broad thermal spectrum (10-60 °C), which can promote high plastic action potential through dynamic environmental temperature fluctuations. This enzyme showed following kinetic parameters: kcat = 6.377 s-1 ± 0.303, Km = 4.219 mM, and Vmax = 24.43 µM/min (against ABTS as substrate). LacMeta almost completely degraded malachite green (50 mg/mL) in only 2 h. Moreover, the enzyme was able to degrade seven dyes from four distinct classes and it respectively achieved 85% and 83% decolorization of methylene blue and trypan blue with ABTS as the mediator. In addition, LacMeta showed potential for the degradation of two thirds of an agricultural fungicide: fentin hydroxide, thus demonstrating its biotechnological aptitude for bioremediation. The results of this study suggest that LacMeta has potential in textile wastewater treatment and that it could help in the bioremediation of other human/environmental toxins such as pesticides and antibiotic compounds belonging to the same chemical classes as the degraded dyes.

Bioremediation of heavy metal-polluted environments by non-living cells from rhizobial isolates.

Rhizosphere bacteria, for example, rhizobia, can play several roles, and one of the most important, the protection of plant roots against toxic conditions and other environmental stresses. In this work, the action of Cu2+ and Cr6+ on cell growth and EPS production of four strains of rhizobia, Rhizobium tropici (LBMP-C01), Ensifer sp. (LBMP-C02 and LBMP-C03), and Rhizobium sp. LBMP-C04, were tested. The results confirmed the strong effect of Cu2+ and Cr6+ on bacterial exopolysaccharides (EPS) synthesis, and how cells can adsorb these metals, which may be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal-contaminated soils. Here, we emphasize the importance of proving the potential of treating bacterial cells and their extracellular EPS to promote the bio-detoxification of terrestrial and aquatic systems contaminated by heavy metals in a highly sustainable, economic, and ecological way.

Metagenome-assembled genome of a Chitinophaga sp. and its potential in plant biomass degradation, as well of affiliated Pandoraea and Labrys species.

The prospection of new degrading enzymes of the plant cell wall has been the subject of many studies and is fundamental for industries, due to the great biotechnological importance of achieving a more efficient depolymerization conversion from plant polysaccharides to fermentable sugars, which are useful not only for biofuel production but also for various bioproducts. Thus, we explored the shotgun metagenome data of a bacterial community (CB10) isolated from sugarcane bagasse and recovered three metagenome-assembled genomes (MAGs). The genomic distance analyses, along with phylogenetic analysis, revealed the presence of a putative novel Chitinophaga species, a Pandoraea nosoerga, and Labrys sp. isolate. The isolation process for each one of these bacterial lineages from the community was carried out in order to relate them with the MAGs. The recovered draft genomes have reasonable completeness (72.67-100%) and contamination (0.26-2.66%) considering the respective marker lineage for Chitinophaga (Bacteroidetes), Pandoraea (Burkholderiales), and Labrys (Rhizobiales). The in-vitro assay detected cellulolytic activity (endoglucanases) only for the isolate Chitinophaga, and its genome analysis revealed 319 CAZymes, of which 115 are classified as plant cell wall degrading enzymes, which can act in fractions of hemicellulose and pectin. Our study highlights the potential of this Chitinophaga isolate provides several plant-polysaccharide-degrading enzymes.

The potential of extracellular biopolymer production by Mesorhizobium sp. from monosaccharide constituents of lignocellulosic biomass.

OBJECTIVE: The effects of monosaccharide constituents of lignocellulosic materials on exopolysaccharide (EPS) production by Mesorhizobium sp. Semia 816 were studied. RESULTS: According to the results, by using sugars commonly found in lignocellulosic biomass as carbon sources (glucose, arabinose and xylose), no significant differences were observed in the production of EPS, reaching 3.39 g/L, 3.33 g/L and 3.27 g/L, respectively. Differences were observed in monosaccharide composition, mainly in relation to rhamnose and glucuronic acid contents (1.8 times higher when arabinose was compared with xylose). However, the biopolymers showed no differences in relation to rheological properties, with EPS aqueous-based suspensions (1.0% w/v) presenting pseudoplastic behavior, and a slight difference in degradation temperatures. Using soybean hulls hydrolysate as carbon source, slightly higher values were obtained (3.93 g/L). CONCLUSION: The results indicate the potential of the use of lignocellulosic hydrolysates containing these sugars as a source of carbon in the cultivation of Mesorhizobium sp. Semia 816 for the production of EPS with potential industrial applications.

Differential Expression of Monocarboxylate Transporter 1 and Ancillary Protein CD147 in Red Blood Cells of Show Jumping Horses.

We compare the expression levels of the lactate transporter complex consisting of the lactate transporter, monocarboxylate transporter 1 (MCT1), and its ancillary protein, cluster of differentiation 147 (CD147), in the membranes of red blood cells (RBCs) from two breeds of jumping horses and associate the expression levels of these proteins with their jumping ability. The expression levels of MCT1 and CD147 proteins on the membranes of RBCs collected from 30 show jumping horses of two different breeds were quantified: the Brazilian Sport Horses (n = 17) and the European Warmbloods (n = 13). The levels of MCT1 and CD147 in the RBC membranes were measured by Western blot using horse-specific antibodies. Statistical analyses included unpaired Student t-test and Chi-squared test. According to the expression levels of MCT1 and CD147 proteins, 88% of the Brazilian Sport Horses were categorized as high lactate transporters and the remaining 12% as low lactate transporters. The opposite was found for the European Warmbloods, where most animals (77%) were classified as low lactate transporters and the remaining animals (23%) were classified as high lactate transporters. Brazilian Sport Horses express statistically significantly higher levels of CD147 and MCT1 than European Warmbloods. The classification of horses considering the expression of proteins involved in the ability to transport lactate through the complex MCT1-CD147 seems to be breed dependent, with horses that are able to jump higher obstacles showing lower expression of the MCT1-CD147 complex in their RBCs.

Rhizobium spp exopolysaccharides production and xanthan lyase use on its structural modification.

Enzymes can be very useful on exopolysaccharides (EPS) research, can be used at elucidation and also to modify the polysaccharides' structure in order to alter their physical properties. Thus, the reduction of the molecular mass could increase applications of these biopolymers. Therefore, the EPS production of different rhizobia and the action of xanthan lyase on its structures were evaluated. The strains produced significant amounts of EPS, and it was noticed that are heteropolysaccharides, composed galactose and glucose. Both EPS and xanthan were modified on ß-glycosidic bonds, the mannose was removed of xanthan had but the EPS was affected in the CO stretching vibration, where the glucuronic acid removed from of your structure. The ester/carboxylic acid portions affected functional groups of the acetate/succinate, methyl carbons of the O-acetyl and pyruvate methyl groups in addition to affect the carbons the main pyranoid. The Resistance to temperature increase of the EPS was observed, made possible by the activity of the lyase. EPS has the ability to form stable gels at higher temperatures and anionic feature can be used on solubilization and controlled release of substances. Modified EPS knowledge will presently facilitate future investigations relating the structure of the rhizobia polysaccharide against rheological properties.

From a metagenomic source to a high-resolution structure of a novel alkaline esterase.

Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/ß hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.

Characterization of new exopolysaccharide production by Rhizobium tropici during growth on hydrocarbon substrate.

Exopolysaccharide (EPS) are produced by a diverse of rhizobia species and has been demonstrated to be a bioemulsifier with potential applications in the degradation of hydrocarbons. In the present study, attempts were made to obtain the new exopolysaccharide production by Rhizobium tropici (SEMIA 4080 and MUTZC3) strains during growth on hydrocarbon substrate. Under the different cultivation conditions, the high molecular weight exopolysaccharides from Rhizobium tropici strains cultivated for 96h mainly consisted of carbohydrates (79-85%) and a low percentage of protein. The EPSC3-D differed from the others, with only 60% of carbohydrate. However, all strains produced polymers with distinct rheology properties, such as viscosity of each EPS sample, suitable for different applications. In addition, RP-HPLC, FTIR and NMR studies revealed EPS produced by rhizobia strains were similar indicating minimal difference between EPS compositions.

Liming in the sugarcane burnt system and the green harvest practice affect soil bacterial community in northeastern São Paulo, Brazil.

Here we show that both liming the burnt sugarcane and the green harvest practice alter bacterial community structure, diversity and composition in sugarcane fields in northeastern São Paulo state, Brazil. Terminal restriction fragment length polymorphism fingerprinting and 16S rRNA gene cloning and sequencing were used to analyze changes in soil bacterial communities. The field experiment consisted of sugarcane-cultivated soils under different regimes: green sugarcane (GS), burnt sugarcane (BS), BS in soil amended with lime applied to increase soil pH (BSL), and native forest (NF) as control soil. The bacterial community structures revealed disparate patterns in sugarcane-cultivated soils and forest soil (R = 0.786, P = 0.002), and overlapping patterns were shown for the bacterial community structure among the different management regimes applied to sugarcane (R = 0.194, P = 0.002). The numbers of operational taxonomic units (OTUs) found in the libraries were 117, 185, 173 and 166 for NF, BS, BSL and GS, respectively. Sugarcane-cultivated soils revealed higher bacterial diversity than NF soil, with BS soil accounting for a higher richness of unique OTUs (101 unique OTUs) than NF soil (23 unique OTUs). Cluster analysis based on OTUs revealed similar bacterial communities in NF and GS soils, while the bacterial community from BS soil was most distinct from the others. Acidobacteria and Alphaproteobacteria were the most abundant bacterial phyla across the different soils with Acidobacteria Gp1 accounting for a higher abundance in NF and GS soils than burnt sugarcane-cultivated soils (BS and BSL). In turn, Acidobacteria Gp4 abundance was higher in BS soils than in other soils. These differential responses in soil bacterial community structure, diversity and composition can be associated with the agricultural management, mainly liming practices, and harvest methods in the sugarcane-cultivated soils, and they can be detected shortly after harvest.

Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications.

Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.

Properties of Polyhydroxyalkanoate Granules and Bioemulsifiers from Pseudomonas sp. and Burkholderia sp. Isolates Growing on Glucose.

A Burkholderia and Pseudomonas species designated as AB4 and AS1, respectively, were isolated from soil containing decomposing straw or sugar cane bagasse collected from Brazil. This study sought to evaluate the capacities of culture media, cell-free medium, and crude lysate preparations (containing PHB inclusion bodies) from bacterial cell cultures to stabilize emulsions with several hydrophobic compounds. Four conditions showed good production of bioemulsifiers (E24 ≥ 50 %), headed by substantially cell-free media from bacterial cell cultures in which bacterial isolates from Burkholderia sp. strain AB4 and Pseudomonas sp. strain AS1 were grown. Our results revealed that the both isolates (AB4 and AS1 strains) exhibited high emulsification indices (indicating usefulness in bioremediation) and good stabilities.

Investigation, expression, and molecular modeling of ORF2, a metagenomic lipolytic enzyme.

One clone exhibiting lipolytic activity was selected among 30 positives from a metagenomic library of a microbe consortium specialized in petroleum hydrocarbon degradation. From this clone, a sublibrary was constructed and a metagenome contig was assembled and analyzed using the ORF Finder; thus, it was possible to identify a potential ORF that encodes a lipolytic enzyme, denoted ORF2. This ORF is composed of 1035-bp 345 amino acids and displayed 98 % identity with an alpha/beta hydrolase from Pseudomonas nitroreducens (accession number WP024765380.1). When analyzed against a metagenome database, ORF2 also showed 76 % of sequence identity with a hypothetical protein from a marine metagenome (accession number ECT55726.1). The ProtParam analyses indicated that the recombinant protein ORF2 has a molecular mass approximately 39 kDa, as expected from its amino acid sequence, and based on phylogenetic analysis and molecular modeling, it was possible to suggest that ORF2 is a new member from family V. This enzyme exhibits the catalytic triad and conserved motifs typical from this family, wherein the serine residue is located in the central position of the conserved motif GASMGG. The orf2 gene was cloned in the expression vector pET28a, and the recombinant protein was superexpressed in Escherichia coli BL21(DE3) cells. The lipolytic activity of protein bands presented in a SDS-PAGE gel was confirmed by zymogram analyses, indicating ORF2 activity. These discoveries raise the possibility of employing this protein in biotechnological applications, such as bioremediation.

Production of exopolysaccharide from rhizobia with potential biotechnological and bioremediation applications.

The potential use of rhizobia under controlled fermentation conditions may result in the production of new extracellular polymeric substances (EPS) having novel and superior properties that will open up new areas of industrial applications and thus increase their demand. The production of EPS and the stability of emulsions formed with soybean oil, diesel oil and toluene using different concentrations of purified EPS derived from wild-type and mutant strains of Rhizobium tropici SEMIA 4077 was investigated. The EPS was defined as a heteropolysaccharide composed of six constituent monosaccharides that displayed higher intrinsic viscosity and pseudoplastic non-Newtonian fluid behavior in an aqueous solution. The ratio between the total EPS production and the cellular biomass was 76.70 for the 4077::Z04 mutant strain and only 8.10 for the wild-type strain. The EPS produced by the wild-type R. tropici SEMIA 4077 resulted in more stable emulsions with the tested toluene than xanthan gum, and the emulsification indexes with hydrocarbons and soybean oil were higher than 50%, indicating strong emulsion-stabilizing capacity. These results demonstrate that the EPS of R. tropici strains could be attractive for use in industrial and environmental applications, as it had higher intrinsic viscosity and good emulsification activity.

Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli.

BACKGROUND: The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. RESULTS: Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 µM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. CONCLUSION: The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops.

Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds.

Vitrification, a simple, fast, and recommended cryopreservation method for orchid germplasm conservation, was evaluated for Dendrobium hybrid "Dong Yai" mature seeds. The genetic stability of regenerated seedlings was also evaluated using flow cytometry. Mature seeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3, 4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at -196 °C for 1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seed germination was evaluated after 30 days. Seeds directly submitted to LN did not germinate after cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the best germination (between 51 and 58%), although longer exposure to PVS2 returned moderated germination (39%). Germinated seeds were further subcultured in P-723 culture medium and developed whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases, or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse conditions with over 80% survival. Flow cytometry analysis revealed no chromosomal changes on vitrified seedlings, as well as seedlings germinated from the control treatment (direct exposure to LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreservation method for commercial Dendrobium orchid hybrid conservation.

Biotechnology of polyketides: new breath of life for the novel antibiotic genetic pathways discovery through metagenomics.

The discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. In fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the "goose that laid the golden egg," the potential of this wealth is still inexorable: simply adjust the focus from "micro" to "nano", that means changing the look from microorganisms to nanograms of DNA. Then, the search for new drugs, driven by genetic engineering combined with metagenomic strategies, shows us a way to bypass the barriers imposed by methodologies limited to isolation and culturing. However, we are far from solving the problem of supplying new molecules that are effective against the plasticity of multi- or pan-drug-resistant pathogens. Although the first advances in genetic engineering date back to 1990, there is still a lack of high-throughput methods to speed up the screening of new genes and design new molecules by recombination of pathways. In addition, it is necessary an increase in the variety of heterologous hosts and improvements throughout the full drug discovery pipeline. Among numerous studies focused on this subject, those on polyketide antibiotics stand out for the large technical-scientific efforts that established novel solutions for the transfer/engineering of major metabolic pathways using transposons and other episomes, overcoming one of the main methodological constraints for the heterologous expression of major pathways. In silico prediction analysis of three-dimensional enzymatic structures and advances in sequencing technologies have expanded access to the metabolic potential of microorganisms.

Genetic diversity and population differentiation of Guignardia mangiferae from "Tahiti" acid lime.

Among the citrus plants, "Tahiti" acid lime is known as a host of G. mangiferae fungi. This species is considered endophytic for citrus plants and is easily isolated from asymptomatic fruits and leaves. G. mangiferae is genetically related and sometimes confused with G. citricarpa which causes Citrus Black Spot (CBS). "Tahiti" acid lime is one of the few species that means to be resistant to this disease because it does not present symptoms. Despite the fact that it is commonly found in citric plants, little is known about the populations of G. mangiferae associated with these plants. Hence, the objective of this work was to gain insights about the genetic diversity of the G. mangiferae populations that colonize "Tahiti" acid limes by sequencing cistron ITS1-5.8S-ITS2. It was verified that "Tahiti" acid lime plants are hosts of G. mangiferae and also of G. citricarpa, without presenting symptoms of CBS. Populations of G. mangiferae present low-to-moderate genetic diversity and show little-to-moderate levels of population differentiation. As gene flow was detected among the studied populations and they share haplotypes, it is possible that all populations, from citrus plants and also from the other known hosts of this fungus, belong to one great panmictic population.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization.

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X. fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with alpha-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed "cyanogenesis", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X. fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X. fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs.

Strain variability in the DNA immigration control region (ICR) of Xylella fastidiosa.

The genome of the bacterium Xylella fastidiosa contains four ORFs (XF2721, XF2725, XF2739 and XF0295) related to the restriction modification type I system, ordinarily named R-M. This system belongs to the DNA immigration control region (ICR). Each ORF is related to different operon structures, which are homologues among themselves and with subunit Hsd R from the endonuclease coding genes. In addition, these ORFs are highly homologous to genes in Pseudomonas aeruginosa, Methylococcus capsulatus str. Bath, Legionella pneumophila, Helicobacter pylori, Xanthomonas oryzae pv. Oryzae and Silicibacter pomeroyi, as well as to genes from X. fastidiosa strains that infect grapevine, almond and oleander plants. This study was carried out on R-M ORFs from forty-three X. fastidiosa strains isolated from citrus, coffee, grapevine, periwinkle, almond and plum trees, in order to assess the genetic diversity of these loci through PCR-RFLP. PCR-RFLP analysis of the four ORFs related to the R-M system from these strains enabled the detection of haplotypes for these loci. When the haplotypes were defined, wide genetic diversity and a large range of similar strains originating from different hosts were observed. This analysis also provided information indicating differences in population genetic structures, which led to detection of different levels of gene transfer among the groups of strains.