Characterization of the abn2(yxiA) encoding a Bacillus subtilis GH43 arabinanase, Abn2, and its role in arabino-polysaccharides degradation.

Journal of Bacteriology (Junho 2008) 4272-4280The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-alpha-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-alpha-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent K(m) of 2.0 mg ml(-1) and V(max) of 0.25 mmol min(-1) mg(-1) at pH 7.0 and 50 degrees C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a sigma(A)-dependent and a sigma(H)-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.This work was partially supported by grant no. POCI/AGR/60236/2004 from Fundacao para a Ciencia e Tecnologia (FCT) and FEDER to I.D.S.-N. and by fellowship SFRH/BD/18238/2004 from FCT to J.M.I

A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis

Journal of Bacteriology (Out 2010) 5312-5318Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.This work was supported partially by grant no. PTDC/AGR-AAM/102345/2008 from Fundacao para a Ciencia e Tecnologia (FCT), FEDER, and POFC 2010, to I.S.-N

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis.

Microbiology 154 (2008) 2719-2729Bacillus subtilis produces alpha-l-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and l-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (>250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 degrees C, with p-nitrophenyl-alpha-l-arabinofuranoside as substrate, gave an apparent K(m) of 0.498 mM and 0.421 mM, and V(max) of 317 U mg(-1) and 311 U mg(-1) for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 degrees C and 60 degrees C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1-->5) linkages of linear alpha-1,5-l-arabinan and alpha-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1-->2) and (1-->3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.This work was partially supported by grant no. POCI/AGR/60236/2004 from the Fundacao para a Ciencia e Tecnologia (FCT) and FEDER to I. d. S.- N., and fellowship SFRH/BD/18238/2004 from the FCT to J. M. I

Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-alpha-arabinanase from Bacillus subtilis.

Acta Crystallographica F64 (2008) 636-638Two Bacillus subtilis extracellular endo-1,5-alpha-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-alpha-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 A, alpha = 82.3, beta = 87.9, gamma = 63.6 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 A. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 A resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.This work was partially supported by grant No. POCI/AGR/60236/2004 from Fundacao para a Ciencia e Tecnologia (FCT) and FEDER to IdS-N and fellowship SFRH/BD/18238/2004 from FCT to JMI

Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon

In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR–DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR–DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability

We would like to thank Herminia de Lencastre (ITQB-AX NOVA), Adriano Henriques (ITQB-AX NOVA) and PaulaTamagnini (i3S) for the gift of chromosomal DNA, Paulo Tavares (I2BC-CNRS) and Adriano Henriques for the gift of flagellin and anti-sigmaA antibodies, respectively, and Diogo Pereira for carrying out some growth kinetic assays. This work was supported by the FundacAo para a Ciencia e a Tecnologia [Grant Numbers PTDC/BIA-MIC/30696/2017 to ISN and UID/Multi/04378/2019 to Unidade de Ciencias Biomoleculares Aplicadas UCIBIO; and fellowship PD/BD/105737/2014 to F.L.].ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.publishersversionpublishe

Integridad de la piel en ancianos: revisión de literatura de acuerdo con las cartas de promoción de la salud

Trata-se de uma revisão bibliográfica que objetivou relacionar as medidas educativas para a promoção da integridade da pele em idosos com as Cartas de Promoção da Saúde. Realizou-se a busca nas bases de dados CINAHL, SCOPUS, LILACS e COCHRANE, nos portais CAPES e BVS e na biblioteca PUBMED, mediante a aplicação dos descritores Health Education; Skin e Aged. Os resultados dos 7 artigos analisados apontaram como principais medidas educativas: inspeção diária da pele, cuidados com calçados e com os pés, uso regular de protetor solar e mudanças de decúbito. Essas medidas estavam relacionadas com as seguintes Cartas de Promoção da Saúde: Ottawa, Declaração de Santafé de Bogotá e Declaração de Jacarta. Conclui-se que as medidas educativas, baseadas nas Cartas, são de grande relevância para a criação de uma cultura de saúde, com enfoque na população e no indivíduo como agentes executores imprescindíveis para o alcance da promoção da saúdeThis bibliographical review aimed to relate educational measures for the promotion of skin integrity in older adults with the Health Promotion Charters. The search was performed in the databases CINAHL, SCOPUS, LILACS and COCHRANE, in the CAPES and BVS portals and in the PUBMED library, using the descriptors Health Education; Skin and Aged. The results from the 7 articles analyzed indicate, as the principle educative measures, daily inspection of the skin, care with footwear and the feet, regular use of sunblock and changes in supine position. These measures were related to the following Charters for Health Promotion: Ottawa, the Declaration of Bogotá and the Jakarta Declaration. It is concluded that educational measures, based on the Charters, are highly relevant for the creation of a culture of health, with focus on the population and individual as essential operative agents for the achievement of health promotionEsta es una revisión bibliográfica cuyo objetivo fue relacionar las medidas educativas para la promoción de la integridad de la piel en ancianos por medio de las Cartas de Promoción de la Salud. La búsqueda fue realizada en las bases de datos CINAHL, SCOPUS, LILACS y COCHRANE, en portales CAPES y BVS y en la biblioteca PUBMED, por medio de la aplicación de los descriptores Health Education; Skin y Aged. Los resultados de los 7 artículos analizados apuntan como principales medidas educativas: inspección no diaria de la piel, cuidados con zapatos y con los pies, uso regular de protector solar y cambios de decúbito. Tales medidas estaban relacionadas con las siguientes Cartas de Promoción de la Salud: Ottawa, Declaración de Santafé de Bogotá y Declaración de Jacarta. La conclusión es de que las medidas educativas, basadas en las Cartas, tienen gran relevancia para la creación de una cultura de salud, con enfoque en la población y en el individuo como agentes ejecutores imprescindibles para llegar a la promoción de la salu

INTEGRIDAD DE LA PIEL EN ANCIANOS: REVISIÓN DE LITERATURA DE ACUERDO CON LAS CARTAS DE PROMOCIÓN DE LA SALUD

Trata-se de uma revisão bibliográfica que objetivou relacionar as medidas educativas para a promoção da integridade da pele em idosos com as Cartas de Promoção da Saúde. Realizou-se a busca nas bases de dados CINAHL, SCOPUS, LILACS e COCHRANE, nos portais CAPES e BVS e na biblioteca PUBMED, mediante a aplicação dos descritores Health Education; Skin e Aged. Os resultados dos 7 artigos analisados apontaram como principais medidas educativas: inspeção diária da pele, cuidados com calçados e com os pés, uso regular de protetor solar e mudanças de decúbito. Essas medidas estavam relacionadas com as seguintes Cartas de Promoção da Saúde: Ottawa, Declaração de Santafé de Bogotá e Declaração de Jacarta. Conclui-se que as medidas educativas, baseadas nas Cartas, são de grande relevância para a criação de uma cultura de saúde, com enfoque na população e no indivíduo como agentes executores imprescindíveis para o alcance da promoção da saúde.This bibliographical review aimed to relate educational measures for the promotion of skin integrity in olderadults with the Health Promotion Charters. The search was performed in the databases CINAHL, SCOPUS, LILACSand COCHRANE, in the CAPES and BVS portals and in the PUBMED library, using the descriptors Health Education;Skin and Aged. The results from the 7 articles analyzed indicate, as the principle educative measures, daily inspection ofthe skin, care with footwear and the feet, regular use of sunblock and changes in supine position. These measures wererelated to the following Charters for Health Promotion: Ottawa, the Declaration of Bogotá and the Jakarta Declaration. Itis concluded that educational measures, based on the Charters, are highly relevant for the creation of a culture of health,with focus on the population and individual as essential operative agents for the achievement of health promotion.Esta es una revisión bibliográfica cuyo objetivo fue relacionar las medidas educativas para la promoción de laintegridad de la piel en ancianos por medio de las Cartas de Promoción de la Salud. La búsqueda fue realizada en las basesde datos CINAHL, SCOPUS, LILACS y COCHRANE, en portales CAPES y BVS y en la biblioteca PUBMED, por mediode la aplicación de los descriptores Health Education; Skin y Aged. Los resultados de los 7 artículos analizados apuntancomo principales medidas educativas: inspección no diaria de la piel, cuidados con zapatos y con los pies, uso regular deprotector solar y cambios de decúbito. Tales medidas estaban relacionadas con las siguientes Cartas de Promoción de laSalud: Ottawa, Declaración de Santafé de Bogotá y Declaración de Jacarta. La conclusión es de que las medidas educativas,basadas en las Cartas, tienen gran relevancia para la creación de una cultura de salud, con enfoque en la población y enel individuo como agentes ejecutores imprescindibles para llegar a la promoción de la salud

Diagnóstico oportuno da tuberculose nos serviços de saúde de diversas regiões do Brasil

OBJETIVO: Analizar el primer contacto del paciente con los servicios de salud para el diagnóstico oportuno de la tuberculosis (TB) en diferentes regiones de Brasil. MÉTODO: Estudio transversal en seis ciudades en el sudeste, sur y noreste. La recolección de datos utilizó fuentes secundarias y entrevistas con los pacientes. Los datos fueron analizados utilizando técnicas descriptivas y el análisis factorial de correspondencias múltiples. RESULTADOS: La Atención Primaria obtuve menor proporción de diagnósticos y mayor tiempo. Los servicios asociados con el diagnóstico en la primera visita fueron los servicios especializados y los Programas de Control de la Tuberculosis, que ofrecen consultas y exámenes in situ. CONCLUSIÓN: Es necesaria la organización del trabajo integrado entre los equipos de los diferentes servicios para las acciones de control. En la Atención Primaria, es necesario también observar el grado de desarrollo y sostenibilidad en la aplicación de estas medidas a la práctica diaria de los servicios.OBJETIVO: analisar o primeiro contato do doente com os serviços de saúde para o diagnóstico oportuno da tuberculose (TB), em diferentes regiões do Brasil. MÉTODO: trata-se de estudo de coorte transversal, em 6 municípios das Regiões Sudeste, Sul e Nordeste. Para a coleta utilizaram-se fontes secundárias e entrevista com os doentes. Os dados foram analisados por meio de técnicas descritivas e análise fatorial de correspondência múltipla. RESULTADOS: a Atenção Primária à Saúde apresentou maior tempo e menor proporção de diagnósticos. Os serviços associados ao diagnóstico, na primeira consulta, foram os serviços especializados e os Programas de Controle da TB, que oferecem consulta e exames no próprio local. CONCLUSÃO: faz-se necessária a organização do trabalho de forma integrada, entre as equipes dos diferentes serviços, para as ações de controle da TB. Na Atenção Primária à Saúde, é preciso ainda observar o grau de incorporação e sustentabilidade na execução dessas ações à prática diária dos serviços.OBJECTIVE: To analyze the infected person's first contact with the health services, for the early diagnosis of tuberculosis (TB) in different regions of Brazil. METHOD: a cross-sectional study undertaken in 6 municipalities in the South-east, South and North-east regions of Brazil. Data collection involved secondary sources and interviews with the patients. The data was analyzed using descriptive techniques and multiple correspondence factor analysis. RESULTS: Primary Health Care (PHC) presented the longest time to diagnosis and the lowest proportion of diagnoses. The services associated with diagnosis in the first consultation were the specialized services and the Tuberculosis Control Programs, which offer consultations and tests on-site. CONCLUSION: For the control of TB, it is necessary to organize the work in an integrated way between the different services' teams. In Primary Health Care, it is also necessary to observe the extent of incorporation and sustainability in the implementation of these actions into the health services' daily practice