Gut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharides.

The largest living rodent, capybara, can efficiently depolymerize and utilize lignocellulosic biomass through microbial symbiotic mechanisms yet elusive. Herein, we elucidate the microbial community composition, enzymatic systems and metabolic pathways involved in the conversion of dietary fibers into short-chain fatty acids, a main energy source for the host. In this microbiota, the unconventional enzymatic machinery from Fibrobacteres seems to drive cellulose degradation, whereas a diverse set of carbohydrate-active enzymes from Bacteroidetes, organized in polysaccharide utilization loci, are accounted to tackle complex hemicelluloses typically found in gramineous and aquatic plants. Exploring the genetic potential of this community, we discover a glycoside hydrolase family of ß-galactosidases (named as GH173), and a carbohydrate-binding module family (named as CBM89) involved in xylan binding that establishes an unprecedented three-dimensional fold among associated modules to carbohydrate-active enzymes. Together, these results demonstrate how the capybara gut microbiota orchestrates the depolymerization and utilization of plant fibers, representing an untapped reservoir of enzymatic mechanisms to overcome the lignocellulose recalcitrance, a central challenge toward a sustainable and bio-based economy.

Trends in biological data integration for the selection of enzymes and transcription factors related to cellulose and hemicellulose degradation in fungi.

Fungi are key players in biotechnological applications. Although several studies focusing on fungal diversity and genetics have been performed, many details of fungal biology remain unknown, including how cellulolytic enzymes are modulated within these organisms to allow changes in main plant cell wall compounds, cellulose and hemicellulose, and subsequent biomass conversion. With the advent and consolidation of DNA/RNA sequencing technology, different types of information can be generated at the genomic, structural and functional levels, including the gene expression profiles and regulatory mechanisms of these organisms, during degradation-induced conditions. This increase in data generation made rapid computational development necessary to deal with the large amounts of data generated. In this context, the origination of bioinformatics, a hybrid science integrating biological data with various techniques for information storage, distribution and analysis, was a fundamental step toward the current state-of-the-art in the postgenomic era. The possibility of integrating biological big data has facilitated exciting discoveries, including identifying novel mechanisms and more efficient enzymes, increasing yields, reducing costs and expanding opportunities in the bioprocess field. In this review, we summarize the current status and trends of the integration of different types of biological data through bioinformatics approaches for biological data analysis and enzyme selection.

"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.

An engineered GH1 ß-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

ß-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of ß-1, 4-glycosidic linkages, ß-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal ß-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 ß-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 ß-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

Solubilization, Folding, and Purification of a Recombinant Peptidoglycan-Associated Lipoprotein (PAL) Expressed in Escherichia coli.

Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL-enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.

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.

Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 ß-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions.

BACKGROUND: The conversion of biomass-derived sugars via enzymatic hydrolysis for biofuel production is a challenge. Therefore, the search for microorganisms and key enzymes that increase the efficiency of the saccharification of cellulosic substrates remains an important and high-priority area of study. Trichoderma harzianum is an important fungus known for producing high levels of cellulolytic enzymes that can be used for cellulosic ethanol production. In this context, ß-glucosidases, which act synergistically with cellobiohydrolases and endo-ß-1,4-glucanases in the saccharification process, are potential biocatalysts for the conversion of plant biomass to free glucose residues. RESULTS: In the present study, we used RNA-Seq and genomic data to identify the major ß-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the ß-glucosidases from glycoside hydrolase families 1 and 3, and we identified the enzyme with the highest expression under these conditions. The target gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein (rThBgl) was purified with high yields. rThBgl was characterized using a comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques. Finally, we determined the crystallographic structure of the recombinant protein at a resolution of 2.6 Å. CONCLUSIONS: Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a ß-glucosidase that is highly expressed by T. harzianum under biomass degradation conditions. The methodology described in this manuscript will be useful for the bio-prospection of key enzymes, including cellulases and other accessory enzymes, for the development and/or improvement of enzymatic cocktails designed to produce ethanol from plant biomass.

The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles.

The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

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.

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.

Identification of oxidoreductases from the petroleum Bacillus safensis strain.

A gram-positive bacterium, denominated CFA-06, was isolated from Brazilian petroleum in the Campos Basin and is responsible for the degradation of aromatic compounds and petroleum aromatic fractions. The CFA-06 strain was identified as Bacillus safensis using the 16S rRNA and gyrase B sequence. Enzymatic assays revealed the presence of two oxidoreductases: a catalase and a new oxidoreductase. The oxidoreductases were enzymatically digested and analyzed via ESI-LTQ-Orbitrap mass spectrometry. The mass data revealed a novel oxidoreductase (named BsPMO) containing 224 amino acids and 89% homology with a hypothetic protein from B. safensis (CFA-06) and a catalase (named BsCat) with 491 amino acids and 60% similarity with the catalase from Bacillus pumilus (SAFR-032). The new protein BsPMO contains iron atom(s) and shows catalytic activity toward a monooxygenase fluorogenic probe in the presence of cofactors (NADH, NADPH and NAD). This study enhances our knowledge of the biodegradation process of petroleum by B. safensis.

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.

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.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.

Highly-sensitive and label-free indium phosphide biosensor for early phytopathogen diagnosis.

The development of highly-sensitive and label-free operating semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an Indium Phosphide (InP) semiconductor-based resistive biosensor using the change of its electronic properties upon biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the procedure of functionalization and covalent biomolecule immobilization was also optimized to guarantee high molecule density and high reproducibility which are prerequisite for reliable results. The characterization, such as biomolecular conjugation efficiency, detection concentration limits, receptor:ligand specificity and concentration detection range was analyzed by using three different biological systems: i) synthetic dsDNA and two phytopathogenic diseases, ii) the severe CB-form of Citrus Tristeza Virus (CTV) and iii) Xylella fastidiosa, both causing great economic loss worldwide. The experimental results show a sensitivity of 1 pM for specific ssDNA detection and about 2 nM for the specific detection of surface proteins of CTV and X. fastidiosa phytopathogens. A brief comparison with other semiconductor based biosensors and other methodological approaches is discussed and confirms the high sensitivity and reproducibility of our InP based biosensor which could be suitable for reliable early infection diagnosis in environmental and life sciences.

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.

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains.

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.