Orientador: Nelson Arno Wulff / Banca: Priscila Alves da Silva / Banca: Andrea Soares da Costa Fuentes / Resumo: O Huanglongbing (HLB) é a doença mais devastadora dos citros. No Brasil, o HLB está associado a presença de "Candidatus" Liberibacter asiaticus (Las) e Ca. L. americanus (Lam), ambas transmitidas pelo psilídeo Diaphorina citri. O sequenciamento e anotação dos profagos SC1 e SC2 no genoma de Las, identificou uma possível bacteriocina do tipo colicina Ia em SC1, e uma possível proteína de imunidade a colicina em SC2. Lam apresenta profagos similares no seu genoma, porém SP2 é degenerado, sem proteína de imunidade. Como houve uma inversão notável na ocorrência de Las em relação à Lam no Brasil, com o predomínio atual de Las, a presença de ambos os profagos pode conferir uma vantagem adaptativa a Las pela atividade da bacteriocina. Adicionalmente, Las tem lisozimas cromossômicas e de profago, que também podem estar envolvidas em competição bacteriana. A incapacidade de cultivo axênico de Las e Lam requer o uso de sistemas heterólogos para estudos funcionais. Genes com potencial envolvimento na biologia do profago e na competição bacteriana, como as colicinas e lisozimas foram avaliados. As lisozimas CLIBASIA_04790 e ED07_RS0204515, obtidas dos isolados de Las 3PA e Las 4PA, e as colicinas SC1_gp060 e lam_678 obtidas de isolados de Las 2PA, Las Poona e Lam São Paulo foram clonadas em vetor de expressão pET28a e superexpressas em E. coli BL21 (DE3). O crescimento destas bactérias, mensurado pelas médias dos valores de absorbância (OD600), mostraram efeito tóxico das construções com... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Huanglongbing (HLB) is the most devastating disease of citrus. In Brazil, HLB is associated with the presence of "Candidatus" Liberibacter asiaticus (Las) and Ca. L. americanus (Lam), both transmitted by psyllid Diaphorina citri. Sequencing and annotation of prophages SC1 and SC2 in the genome of Las, reveals the presence of a putative bacteriocin, colicin Ia in SC1, while in SC2 a possible colicin immunity-protein was found. Lam has similar prophages in the genome, though SP2 is degenerated, without immunity gene. As there was a striking inversion in the prevalence of Las in detriment of Lam, the presence of both prophages may confer an adaptative advantage to Las, due to the activity of the bacteriocin. Additionally, Las encodes both a chromosomal and phage lysozyme that may also be involved in bacterial competition. The inability of axenic cultivation of Las and Lam requires the use of heterologous systems for functional studies. Genes with potential involvement in prophage biology and bacterial competition, such as colicins and lysozymes, were evaluated. The lysozymes ORFs CLIBASIA_04790 and ED07_RS0204515, from Las isolates 3PA and Las 4PA, and the colicins SC1_gp060 and lam_678 obtained from isolates Las 2PA, Las Poona and Lam São Paulo were cloned into expression vector pET28a and superexpressed in E. coli BL21 (DE3) cells. The growth rate of these bacteria, measured by means of absorbance values (OD600), showed toxic effect of transformants expressing lysozymes, where... (Complete abstract click electronic access below) / Mestre

Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Bioquímica / La microcina E492 es un antibiótico peptídico de 7,9 kDa producido por Klebsiella pneumoniae RYC492 que mediante la formación de canales iónicos produce la despolarización de la membrana citoplasmática de bacterias gram negativo como E. coli, Salmonella, Citrobacter, Enterobacter y Klebsiella. Mediante estudios de clonamiento y expresión en E. coli se estableció que los determinantes genéticos implicados en la producción de microcina activa están contenidos en un segmento de 13 Kpb ubicado en el cromosoma de Klebsiella pneumoniae RYC492. La microcina E492 es exportada por un sistema de transporte de tipo I constituido por un transportador ABC (MceG), una proteína accesoria (MceH) y una proteína de membrana externa (TolC). El sistema de exportación de microcina E492 y el sistema de exportación de colicina V comparten alrededor de un 90% de similitud entre ellos. Estudios preliminares indicaron que el sistema de exportación de colicina V no reemplaza la función del sistema de exportación de microcina E492, y que la especificidad del transporte estaba relacionada con la presencia del gen mceF. El objetivo central de esta tesis consistió en estudiar los mecanismos de especificidad del sistema I de exportación para lo cual se usará como modelo los exportadores que permiten la secreción de colicina V y microcina E492. Mediante estudios de mutagénesis se determinó que MceF es prescindible en la exportación de microcina E492, y que más bien este producto génico participaría en la regulación de esta bacteriocina. Así, se determinó que MceF regula negativamente la expresión de la inmunidad de la microcina E492 y, posiblemente a través de este mecanismo, la expresión de la microcina E492. Para estudiar los determinantes de la especificidad de la exportación se clonaron los sistemas de exportación de microcina E492 (mceHG) y colicina V (cvaAB). Se determinó que el sistema de exportación de colicina no complementa la exportación de microcina E492. Mediante la construcción de sistemas de exportación híbridos entre las proteínas ABC y accesoria de los sistemas de microcina E492 y colicina V se estableció que el transportador de colicina V (CvaB) no permite la exportación de microcina E492. La construcción de transportadores quimeras entre los dominios del transportador de colicina V y microcina E492 permitió establecer que el dominio ABC del sistema de exportación de colicina V determina la exportación específica de su bacteriocina. Para entender los mecanismos moleculares de los determinantes de especificidad se generaron modelos 3D del transportador de microcina E492. Este modelo ayudó a predecir la importancia del residuo D121 en el procesamiento del péptido líder de la microcina E492, lo cual se comprobó experimentalmente. El modelo de MceG también permitió establecer que las diferencias entre los dominios ABC de los transportadores de microcina E492 y colicina V se localizan en una región específica. El modelo 3D también permitió entender la importancia de los cuatro últimos aminoácidos del transportador en el proceso de exportación, lo cual fue verificado experimentalmente. Esta región interactúa directamente con el nucleótido, elemento indispensable para energizar el sistema. Finalmente, se estudió mediante western blot de extractos celulares totales y extractos extracelulares de mutantes en los dominios peptidasa y ABC, el acoplamiento que existe entre el procesamiento de la bacteriocina y su exportación. Estos estudios indicaron que el procesamiento ocurre concomitante con la exportación y que el dominio ABC es necesario para activar a la peptidasa / Microcin E492 is peptide antibiotic of 7,9 kDa produced by Klebsiella pneumoniae RYC492 that through the formation of ion channels produces depolarization of cytoplasmatic membrane of gram-negative strains such as E. coli, Salmonella, Citrobacter, Enterobacter and Klebsiella. Through the cloning and expression in E. coli has been established that the genetic determinants involved in microcin E492 production are encoded in a 13 Kpb DNA segment from the chromosome of K. pneumoniae RYC492. Microcin E492 is secreted by a type I exporter system. This system is formed by an ABC transporter (MceG), an accessory protein (MceH), and the outer membrane protein TolC. The microcin E492 exporter system has more than 90% of similarity with the colicin V system. Preliminary studies showed that colicin V system is unable to replace the function of microcin E492 exporter, and this specificity was related to the mceF gene. The main goal of this thesis was to study the mechanisms of specificity in type I protein export system using as models colicin V and microcin E492 exporter systems. The use of mutants in MceF allowed to establish that MceF was not necessary to export microcin E492, and that this protein would participate in the regulation of microcin E492 expression. Thus, it was established that microcin E492 immunity is negatively regulated by MceF, and probably through this mechanism the expression of microcin E492 could be controlled. To study the elements that are involved in the specificity of export, the microcin E492 (mceHG) and colicin V (cvaAB) exporter systems were cloned. The colicin V exporter system was unable to complement the export of microcin E492. Through the construction of a hybrid between the accessory and exporter proteins of microcin E492 and colicin V exporter systems it was established that CvaB was unable to export microcin E492. The construction of chimeric proteins between the domains of microcin E492 and colicin V exporters showed that the ABC domain of colicin V exporter confers the specificity in the exportation of this bacteriocin. To understand the molecular mechanism of the specificity, 3D models of microcin E492 exporter were generated. These models helped to determine the importance of D121 residue in the processing of microcin E492 leader peptide. The 3D model of MceG allowed locate the region where the differences between the ABC domain of colicin V and microcin E492 exporters reside. The 3D model also permitted to assess the importance of MceG Cterminal region in the processing and export of microcin E492. This region also interacts directly with the nucleotide, a key step to energize the system. Finally, the coupling between microcin E492 processing and export was analyzed through western blot using intracellular and extracellular extracts of mutants in the ABC or peptidase domains. These studies showed that ABC domain is necessary to activate the peptidase

Submitted by Michele Fernanda Souza Dutra (michele.084@terra.com.br) on 2018-09-25T14:24:54Z No. of bitstreams: 1 Dissertação_Mestrado_Michele_FSD_Versão_Final_25.09.18.pdf: 2825020 bytes, checksum: e2a62db2a8d1b14c59646518fe58e8e9 (MD5) / Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-09-26T12:15:01Z (GMT) No. of bitstreams: 1 dutra_mfs_me_araiq_int.pdf: 2808516 bytes, checksum: dbe9ed3db32fc012b5508b3b44d69e43 (MD5) / Made available in DSpace on 2018-09-26T12:15:01Z (GMT). No. of bitstreams: 1 dutra_mfs_me_araiq_int.pdf: 2808516 bytes, checksum: dbe9ed3db32fc012b5508b3b44d69e43 (MD5) Previous issue date: 2018-08-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O Huanglongbing (HLB) é a doença mais devastadora dos citros. No Brasil, o HLB está associado a presença de "Candidatus" Liberibacter asiaticus (Las) e Ca. L. americanus (Lam), ambas transmitidas pelo psilídeo Diaphorina citri. O sequenciamento e anotação dos profagos SC1 e SC2 no genoma de Las, identificou uma possível bacteriocina do tipo colicina Ia em SC1, e uma possível proteína de imunidade a colicina em SC2. Lam apresenta profagos similares no seu genoma, porém SP2 é degenerado, sem proteína de imunidade. Como houve uma inversão notável na ocorrência de Las em relação à Lam no Brasil, com o predomínio atual de Las, a presença de ambos os profagos pode conferir uma vantagem adaptativa a Las pela atividade da bacteriocina. Adicionalmente, Las tem lisozimas cromossômicas e de profago, que também podem estar envolvidas em competição bacteriana. A incapacidade de cultivo axênico de Las e Lam requer o uso de sistemas heterólogos para estudos funcionais. Genes com potencial envolvimento na biologia do profago e na competição bacteriana, como as colicinas e lisozimas foram avaliados. As lisozimas CLIBASIA_04790 e ED07_RS0204515, obtidas dos isolados de Las 3PA e Las 4PA, e as colicinas SC1_gp060 e lam_678 obtidas de isolados de Las 2PA, Las Poona e Lam São Paulo foram clonadas em vetor de expressão pET28a e superexpressas em E. coli BL21 (DE3). O crescimento destas bactérias, mensurado pelas médias dos valores de absorbância (OD600), mostraram efeito tóxico das construções com lisozimas, sendo que clones de E. coli BL21 (DE3) expressando CLIBASIA_04790 (pMD15) e ED07_RS0204515 (pMD17) cresceram cerca de 60 e 70% menos, respectivamente, quando comparados aos controles. Ensaios de SDS-PAGE e Western Blot com anticorpo anti-His6X, evidenciaram a presença de ambas as proteínas na fração insolúvel, de 20 kDa (pMD15) e 12 kDa (pMD17), confirmando a expressão das lisozimas. Para as colicinas, somente o clone expressando SC1_gp060 proveniente do isolado Poona (pMD16) mostrou efeito em E. coli BL21 (DE3), com redução de 70% no crescimento quando comparado aos controles. Estes resultados auxiliam a desvendar a relação entre genes com potencial vantagem adaptativa e seu envolvimento na biologia das Liberibactérias e podem justificar seu uso biotecnológico em plantas geneticamente modificadas contra o HLB. / Huanglongbing (HLB) is the most devastating disease of citrus. In Brazil, HLB is associated with the presence of "Candidatus" Liberibacter asiaticus (Las) and Ca. L. americanus (Lam), both transmitted by psyllid Diaphorina citri. Sequencing and annotation of prophages SC1 and SC2 in the genome of Las, reveals the presence of a putative bacteriocin, colicin Ia in SC1, while in SC2 a possible colicin immunity-protein was found. Lam has similar prophages in the genome, though SP2 is degenerated, without immunity gene. As there was a striking inversion in the prevalence of Las in detriment of Lam, the presence of both prophages may confer an adaptative advantage to Las, due to the activity of the bacteriocin. Additionally, Las encodes both a chromosomal and phage lysozyme that may also be involved in bacterial competition. The inability of axenic cultivation of Las and Lam requires the use of heterologous systems for functional studies. Genes with potential involvement in prophage biology and bacterial competition, such as colicins and lysozymes, were evaluated. The lysozymes ORFs CLIBASIA_04790 and ED07_RS0204515, from Las isolates 3PA and Las 4PA, and the colicins SC1_gp060 and lam_678 obtained from isolates Las 2PA, Las Poona and Lam São Paulo were cloned into expression vector pET28a and superexpressed in E. coli BL21 (DE3) cells. The growth rate of these bacteria, measured by means of absorbance values (OD600), showed toxic effect of transformants expressing lysozymes, whereas E. coli BL21 (DE3) clones expressing CLIBASIA_04790 (pMD15) and ED07_RS0204515 (pMD17) grew about 60 and 70% when compared to controls. SDS-PAGE and Western Blot assays were performed with anti-His6X antibody, showing the presence of both proteins in the insoluble fraction, with 20 kDa (pMD15) and 12 kDa (pMD17), confirming lysozyme expression. In the case of the colicin, only the clone expressing SC1_gp060 from Las Poona strain (pMD16) showed toxic effect, showing reduction of 70% in growth rates when compared to the controls. These results help to unravel the relationship between genes with potential adaptive advantage and their involvement in the biology of Liberibacteria and may justify their biotechnological use in genetically modified plants against HLB. / CNPq: 830857/1999-0 -