Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

<p>Abstract</p> <p>Background</p> <p>Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a <it>Thermus </it>sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.</p> <p>Results</p> <p>Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the <it>tspgwi </it>gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the <it>Thermus </it>sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from <it>Thermus </it>sp.) are dependent on the presence of divalent cations.</p> <p>Conclusion</p> <p>TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.</p

Breakdown of keratin-laden biomass waste by the thermophilic bacterium Fervidobacterium pennivorans strain T

Developing a more sustainable agro-industry has become a necessity in light of the current environmental crisis. Biocatalysts are already adopted in many industrial applications and have quickly optimized, and in some cases replaced, existing biochemical reactions within the modern agro-industry. Extremozymes, in particular, are valuable tools for processes requiring harsh industrial conditions where, for example, increased temperature may be beneficial for the bioavailability and solubility of organic compounds as well as for improvement in degradation of substrates. In this regard, alternatives to landfill disposal or incineration of keratinous materials such as feathers, wool, hides, hair etc. are emerging and efforts in exploiting thermo-stable keratinolytic biocatalysts have been attempted. Nonetheless, keratin degradation remains a complex process poorly understood and thus limiting the current toolbox of useful enzymes and organisms needed to meet all demands. In this study, a newly isolated strain of an anaerobic, thermophilic microorganism belonging to the Thermotogae phylum, Fervidobacterium pennivorans strain T, was assessed for its capability of degrading native chicken feathers. By following a multiomics approach, its proteolytic system was explored in the attempt to isolate new keratinase candidates. First, the physiology of F. pennivorans strain T was further investigated in batch cultures and the first growth curve of an organism of this species was described, showing a generation time of 150 minutes and a long stationary phase. Then, the complete genome of the organism was sequenced and analysed, revealing interesting molecular features, such as inverted genomic blocks, when compared to its most closely related organisms: F. pennivorans DSM9078T and F. islandicum AW-1. The strain T genome was slightly shorter (2002515 base pair) and had ANI values of 97.65 % and 80.90% to the compared organisms, respectively, but the same number of predicted protease-encoding genes (55) were found by gene mining analysis. Next, feather degradation by the organism was up-scaled using a bioreactor to further evaluate its potential in industrial applications and cells were sampled for transcriptomics purposes. F. pennivorans strain T performed mediocrely in the fermenter, but RNA extraction was, however, not successful. From secretomics analysis of growing cultures, an extracellular serine protease named Peg_1025 was identified, showing high sequence conservation with the subtilisin type proteases, especially with subtilisin Ak1 from Geobacillus stearothermophilus strain AK1. By multiple sequencing alignment, the catalytic triad His, Asp, Ser, as well as a signal peptide and a propeptide domain were predicted. Three dimensional structural modelling using subtilisin Ak1 as template, showed Peg_1025 to possess several insertions of unknown functions compared to subtilisin Ak1, only one conserved Ca2+ binding site as well as lack of a disulphide bond in the active cleft. Nonetheless, important structural motifs remained conserved. The enzyme was successfully expressed in E. coli using N- and C-terminal His-tag and soluble proteins were active at 70°C in proteolytic activity assays that used casein as substrate. Phylogenetic analyses revealed that Peg_1025 belongs to a distinct clade of Thermotogae peptidases separated from fervidolysin and Ak1, and as such, it represents the first characterized member of this phylogenetic group. Although the specific role of the serine protease in feather degradation remains unclear, the general results from this study confirm that F. pennivorans strain T possesses a complex machinery with keratinolytic power. The biology of this extremophile remains an intriguing field of exploration, further encouraged by its biotechnological potential that is still left to unfold.Master's Thesis in BiologyBIO399MAMN-BI

FtsH protease and ClpG disaggregase confer fitness advantages to the worldwide prevalent Pseudomonas aeruginosa clone C

Pseudomonas aeruginosa is an environmental bacterium and a frequent nosocomial pathogen causing a wide range of opportunistic infections, especially in immunocompromised patients. Clone C is one of the most prevalent groups of closely related strains distributed worldwide in the environment, such as in natural aquatic habitats, and the clinical settings, such as in patients with an underlying functional impairment of the cystic fibrosis transmembrane conductance regulator. Clone C strains specifically harbor the horizontally-transferred genomic island PACGI-1. One border of the genomic island contains the transmissible locus of protein quality control (TLPQC), alternatively called the Locus of Heat Resistance (LHR) in other bacteria, predominantly encoding stress-related gene products such as proteins involved in proteostasis. Among those gene products is a xenolog of the gene encoding the AAA+ (ATPase associated with diverse cellular activities) membrane-bound protease FtsH termed FtsH2 and the AAA+ disaggregase ClpG termed ClpGGI. In clone C isolates, ftsH2 and clpGGI encoded on TLPQC exist in addition to the core genome homologs ftsH1 and clpG. This thesis investigates the divergent and convergent roles of the genomic island and core genome copies of ftsH and clpG in fitness and prevalence of the aquatic clone C isolate P. aeruginosa SG17M. Paper I identifies ftsH1 as a pleiotropic gene in P. aeruginosa SG17M, affecting a multitude of phenotypes related to fitness and adaptation such as growth, motility, biofilm formation, antibiotic resistance, autolysis, secondary metabolite secretion and oxidative and heat shock stress. In the absence of ftsH1, the TLPQC locus copy ftsH2 backs up ftsH1 functionality. FtsH1 and FtsH2 share highly conserved functional AAA+ ATPase and protease domains and form homo- and hetero-oligomers with FtsH2 distinctively produced in the late stationary phase. However, mainly FtsH1 controls the levels of the heat-shock transcription factor RpoH (σ32). Using FtsH trap variants in an in vivo crosslinking/in vitro pull-down experiment shows that the phenazine biosynthesis protein PhzC is a novel substrate for FtsH1 in P. aeruginosa SG17M. Paper II investigates the molecular basis of the differential in vivo functionality of FtsH1 and FtsH2 in P. aeruginosa SG17M. We show that the N-terminal 151 amino acids of FtsH1 are required for FtsH1 functionality and, consequently, optimal growth of P. aeruginosa SG17M. The periplasmic domain and the short glycine-rich cytoplasmic linker connecting the Nterminus to the AAA+ module are particularly crucial for the optimal functionality of FtsH1. Moreover, in vitro biochemical analysis of the purified FtsH proteases shows that FtsH1 and FtsH2 are homo-hexamers and active ATPases with differential degradation activity towards model substrates such as FITC-casein and Arc-st11-ssrA. Paper III studies the role of the ClpG/ClpGGI disaggregases in protein quality control and thermotolerance of P. aeruginosa SG17M. ClpG-type disaggregases confer superior heat tolerance through their high basal ATPase activity coupled to an efficient disaggregase activity. In addition, ClpG/ClpGGI bind aggregates independently without the involvement of the co-chaperone system via a unique N-terminal extension, which contrasts the established ClpB/DnaK co-chaperone system. Paper IV describes the role of the TLPQC/LHR encoding dna-shsp20GI-clpGGI operon in thermotolerance in the human commensal E. coli Fec10 isolate, a close homolog of E. coli K- 12. The horizontally acquired heat tolerance locus, in particular ClpGGI, is a major determinant of tolerance to a lethal temperature upshift to 65 ºC. Biochemically, ClpGGI robustly disaggregates heat-denatured model substrates such as malate dehydrogenase (MDH) and firefly luciferase without the aid of co-chaperone factors. Moreover, ClpGGI shows high intrinsic basal ATPase activity and superior thermal stability compared to the ClpB disaggregase. Paper V reports on the instant double crossover recombination frequencies upon suicide vector integration into chromosomes of the most prevalent P. aeruginosa clone C and PA14 strains. As a result, the genomic engineering of these prevalent clones can be facilitated by omitting the counterselection step. Altogether, two AAA+ proteins, the FtsH protease and the ClpG disaggregases, encoded on the clone C specific genomic island PACGI-1/TLPQC and the core genome, contribute to proteostasis and confer general fitness advantages to the worldwide prevalent P. aeruginosa clone C

Non-destructive control in cheese processing: Modelling texture evolution in the milk curdling phase by laser backscattering imaging

[EN] This study aim was to explore the laser backscattering imaging technique's capacity to model the curdling phase in cheese processing. To do so, three different formulas were studied by modifying solute concentration. Textural modifications to the matrix during curdling were characterised by viscosimetry and texture measurements depending on samples' liquid or solid state. This state changed by determining gelation to establish the limits for the liquid and solid phases. The process was also characterised by the imaging technique, which showed dependence on both solute concentration and enzymatic effect on both the previously observed phases. After following multivariate statistical procedures to reduce dimensionality, the imaging results revealed that solute concentration strongly influenced the variance that the imaging technique captured. It reduced the visibility of the phase change in the image parameters. After eliminating this influence, the evolution of the matrix across the liquid and solid phases was modelled. Data were divided into phases and used to successfully predict the matrix status in each phase by multivariate non-linear regression procedures. It was concluded that the laser backscattering imaging technique presented suitable properties to be used for non-destructive continuous curdling process monitoring during the cheese-making process.The authors gratefully acknowledge the financial support from the University Polytechnic of Valencia for Programme "Ayudas para la Contratacion de Doctores para el Acceso al Sistema Espanol de Ciencia, Tecnologia e Innovacion, en Estructuras de Investigacion de la UPV (PAID-10-17)"Verdú Amat, S.; Pérez Jiménez, AJ.; Barat Baviera, JM.; Grau Meló, R. (2021). Non-destructive control in cheese processing: Modelling texture evolution in the milk curdling phase by laser backscattering imaging. Food Control. 121:1-9. https://doi.org/10.1016/j.foodcont.2020.107638S1912

The architectural complexity of the human PDC core assembly

The mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly linking the glycolytic pathway to the TCA cycle via the specific conversion of pyruvate to acetyl CoA and, as such, is responsible for the maintenance of glucose homeostasis in humans. PDC comprises a central pentagonal dodecahedral core of 60 dihydrolipoamide acetyltransferase (E2) and 12 E3 binding protein (E3BP) subunits. Presently, two conflicting models of PDC (E2+E3BP) core organisation exist: the ‘addition’ (60+12) and ‘substitution’ (48+12) models. In addition to its catalytic role, the multi-domain E2/E3BP core provides the structural framework to which 30 pyruvate decarboxylase (E1) heterotetramers and 6-12 dihydrolipoamide dehydrogenase (E3) homodimers are proposed to bind at maximal occupancy. The formation of specific E2:E1 and E3BP:E3 subcomplexes are characteristic of eukaryotic PDCs and are critical for normal complex function. Despite the availability of limited structural data, the exact subunit organisation and mechanism of operation of the mammalian E2/E3BP core remains unknown. This thesis describes the large-scale purification of tagged, recombinant human PDC cores, full-length rE2 and rE2/E3BP, truncated E2/E3BP, peripheral rE3 enzyme as well as native E2/E3BP core (bE2/E3BP) purified from bovine heart. The ability to purify large amounts of pure protein has enabled the characterisation of the individual cores as well as the E2/E3BP:E3 complex using a variety of biochemical and biophysical techniques. Full-length rE2/E3BP, rE2, bE2/E3BP, truncated E2/E3BP (tLi19/tLi30) and rE2/E3BP:E3 were analysed in solution by analytical ultracentrifugation (AUC). While AUC of the cores supported the substitution model of core organisation, the stoichiometry of interaction was determined to be 2:1 (rE2/E3BP:E3). This was further complemented by gel filtration chromatography (GFC) and small angle neutron scattering (SANS), implying the possible existence of a network of E3 ‘cross-bridges’ linking pairs of E3BP molecules across the surface of the E2 core assembly. Low resolution solution structures obtained for rE2/E3BP, bE2/E3BP and tLi19/tLi30 by small angle x-ray scattering (SAXS) and SANS revealed the presence of icosahedral cores with open pentagonal faces favouring the substitution model of core organisation. These solution structures also indicated high structural similarity between the recombinant and native cores, as well as with the crystal structure obtained previously for the truncated bacterial E2 core. In addition, homology modelling and superimpositions of high- and low-resolution structures of the core revealed conservation of the overall pentagonal dodecahedral morphology despite evolutionary diversity. Evidence for the substitution model of core organisation was further substantiated by negative stain EM of the recombinant and bovine E2/E3BP cores. SANS stoichiometry data indicated the binding of 10 E3 dimers per E2/E3BP core. Although this could correspond to approximately 1:1 stoichiometry between E2/E3BP:E3, subsequent radiolabelling studies suggested possible variation in core subunit composition between the native and recombinant E2/E3BP cores. Therefore, as opposed to the 48E2+12E3BP substitution model based on AUC and SAXS studies with the recombinant E2/E3BP core, rE2/E3BP cores produced in this study indicated a higher level of incorporation of E3BPs with a maximum core composition of 40E2+20E3BP. On the basis of this new finding we have proposed the ‘variable E3BP substitution model’, wherein the number of E3BPs within the core can range from 0 to a maximum of 20, thus resulting in variable populations of E2/E3BP cores. Despite this core variability, the highly controlled regulatory mechanisms in vivo may bias the core composition towards an average of 48E2+12E3BP. However, as the over-expression of the recombinant E2/E3BP core in our study is not as tightly regulated as in vivo, higher number of E3BPs (>12) is observed to be integrated into the core. This new level of architectural complexity and variable subunit composition in mammalian PDC core organisation is likely to have important implications for the catalytic mechanism, overall complex efficiency and tissue-specific regulation by the intrinsic PDC kinases (PDKs) in normal and disease states. The E2 cores of the PDC family are known to be highly flexible, exhibiting inherent size variability reflective of the ‘breathing’ of the core. Integration of E3BP into the E2 core assembly would then be expected to have significant consequences for the structural assembly, affecting the ‘breathing’ and in turn the function and regulation of the complex. Unfolding studies to assess core stability via circular dichroism (CD) and tryptophan fluorescence revealed lower stability of the rE2/E3BP core as compared to cores composed exclusively of rE2 subunits, thus implying the contribution of E3BP towards core destabilisation. In addition, crosslinking studies indicated weak dimerisation of rE3BP, which may be a key factor promoting core destabilisation. The lower stability of the E2/E3BP core may be of benefit in mammals where sophisticated fine tuning is required to obtain cores with optimal catalytic and regulatory efficiencies. SAXS solution structures of E2/E3BP cores obtained were unable to locate the exact positions of E3BP within the core. However, SANS in combination with contrast matching of selectively deuterated components as well as cryo-EM, EM tomography and single molecule studies could be used in future for determination of the exact locations of E3BP, and validating the importance of E2/E3BP core organisation and subunit composition for overall PDC function and regulation

Fatores moleculares associados com a patogenicidade de Phytophthora cinnamomi

Phytophthora cinnamomi is soil pathogen that has a wide range of hosts in several countries and different climates. This fungus is responsible for the chestnut ink disease (Castanea sativa Miller) and death of the tree. Portugal stands out in the production of the European chestnut tree. However, between 2002 and 2004, there was a decrease of 27.3% in the distribution area of this tree due to P. cinnamomi. The aim of this study was to identify molecular factors possibly associated with the fungal pathogenicity through genomic sequences deposited at NCBI using bioinformatics tools. The first contig was used and the OFRs present in de sequences were identified. SmartBlast was used for homologous proteins. The prediction of cellular localization prediction of proteins was performed using four different tools: SignalP 4.1, Cello v.2.5, LOCTree3, Euk-mPLoc 2.0. Protein domains characterization was accomplished using PROSITE and the structure prediction was performed using Phyre2 server. We found 13 proteins probably associated with the pathogenicity of P. cinnamomi and its properties related to the infection were analyzed in silico. These results are important since they are a first step in the search for pathogenic factors.Phytophthora cinnamomi é um patógeno do solo que possui uma ampla gama de hospedeiros em diversos países de diferentes climas. Esse fungo é responsável pela doença da tinta do castanheiro europeu (Castanea sativa Miller) e conduz à morte da árvore. Portugal se destaca na produção do castanheiro europeu. Entretanto, entre 2002 e 2004, houve uma redução de 27.3% na área de distribuição dessa área devido ao P. cinnamomi. O objetivo deste trabalho foi identificar fatores moleculares possivelmente associados à patogenicidade do fungo através de sequências genômicas depositadas no NCBI utilizando ferramentas de bioinformática. O primeiro contig foi utilizado e as ORFs presentes nas sequências foram identificadas. Foi utilizado o smartBlast em busca de proteínas homólogas. A predição da localização celular das proteínas foi feita através de quatro ferramentas: SignalP 4.1, Cello v.2.5, LOCTree3, Euk-mPLoc 2.0. A caracterização dos domínios dos produtos foi realizada utilizando o PROSITE e a predição das estruturas foi realizada utilizando o servidor Phyre2. Foram encontradas 13 proteínas provavelmente associadas com a patogenicidade de P. cinnamomi e as suas propriedades relacionadas com a infecção foram analisadas in silico. Esses resultados são importantes uma vez que são uma primeira etapa na busca por fatores patogênicos.The authors are grateful to Polytechnic Institute of Bragança (IPB) and especially to the Agricultural College of Bragança (IPB) for providing support to carry out this work.info:eu-repo/semantics/publishedVersio

Accessing new biomedical applications by combining genetic design and chemical modification of elastin-like recombinamers

El objetivo de esta tesis es demostrar que la versatilidad de estos recombinámeros se puede aumentar mediante modificación química y genética para la configurar la degradación, el autoensamblado y la interacción con células de los ELRs. El trabajo desarrollado en esta tesis aborda todo el proceso de diseño, producción, purificación, caracterización y aplicación directa de los nuevos ELRs. Para ello, se han utilizado una amplia variedad técnicas de ingeniería genética, microbiología, física, química junto con los correspondientes cultivos celulares. A) La tecnología del ADN recombinante permite un control total sobre el diseño de ELR, y de este modo la inserción de distintas secuencias biofuncionales, como secuencias sensibles a proteasas. Mediante el control de la disposición espacial de este tipo de secuencias proteolíticas queremos demostrar la biodegradación especifica de ELRs. Además, la capacidad de biodegradación selectiva será aplicada para la biofabricación de sustratos para detección zimográfica. B) Debido a la degradabbilidad controlable de los ELRs, su uso será estudiado como sustrato selectivo para la identificación de enzimas proteolíticas. Por lo tanto, siguiendo el estudio de la aplicación de ELRs para técnicas zimográficas diseñaremos un nuevo método de detección de proteasas con potencial para sistemas de inspección avanzados de alto rendimiento. C) Se ha demostrado que los biomateriales modificados con colesterol exhiben fuertes interacciones intermoleculares. Así, aplicaremos estas interacciones en un sistema de ELRs para generar fuerzas intermoleculares que desencadenen el autoensamblado de los ELR. D) Además, gracias a la capacidad de interacción de los grupos colesterol con membranas lipídicas, estudiaremos la capacidad de ELRs ricos en colesterol para mejorar la interacción de los éstos con ciertos tipos celulares implicados en la captación de lípidos, para aumentar el recubrimiento de células vivas con proteínas ELR.Departamento de Física de la Materia Condensada, Cristalografía y MineralogíaDoctorado en Química: Química de Síntesis, Catálisis y Materiales Avanzado