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Correia, IL, Franco IS, de Sá-Nogueira I.  2014.  Towards Novel Amino Acid-Base Contacts in Gene Regulatory Proteins: AraR – A Case Study, 11. PLoS ONE. 9:e111802., Number 11: Public Library of Science AbstractWebsite

AraR is a transcription factor involved in the regulation of carbon catabolism in Bacillus subtilis. This regulator belongs to the vast GntR family of helix-turn-helix (HTH) bacterial metabolite-responsive transcription factors. In this study, AraR-DNA specific interactions were analysed by an in vitro missing-contact probing and validated using an in vivo model. We show that amino acid E30 of AraR, a highly conserved residue in GntR regulators, is indirectly responsible for the specificity of amino acid-base contacts, and that by mutating this residue it will be possible to achieve new specificities towards DNA contacts. The results highlight the importance in DNA recognition and binding of highly conserved residues across certain families of transcription factors that are located in the DNA-binding domain but not predicted to specifically contact bases on the DNA. These new findings not only contribute to a more detailed comprehension of AraR-operator interactions, but may also be useful for the establishment of a framework of rules governing protein-DNA recognition.

McVey, CE, Ferreira MJ, Correia B, Lahiri S, de Sanctis D, Carrondo MA, Lindley PF, de Sá Nogueira I, Soares CM, Bento I.  2014.  The importance of the Abn2 calcium cluster in the endo-1,5-arabinanase activity from Bacillus subtilis. JBIC Journal of Biological Inorganic Chemistry. 19:505-513., Number 4-5: Springer Berlin Heidelberg Abstract

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic β-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.

dos Santos, R, Rocha A, Matias A, Duarte C, de Sá-Nogueira I, Lourenco N, Borges JP, Vidinha P.  2013.  Development of antimicrobial Ion Jelly fibers. RSC Adv.. 3:24400-24405.: The Royal Society of Chemistry Abstract

We report a method to obtain electrospun fibers based on ionic liquids and gelatin, exhibiting antimicrobial properties.

Fontes, CMGA, Correia ISá, Romão MJ, Sá-Nogueira I, Prates JAM, Ferreira LMA.  2012.  Editorial of the 9th Carbohydrate Bioengineering Meeting (CBM9).. Biocatalysis and Biotransformation. 30:273-273., Number 3
Godinho, LM, de Sá-Nogueira I.  2011.  Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis. FEBS Journal. 278:2511–2524., Number 14 Abstract

AraL from Bacillus subtilis is a member of the ubiquitous haloalkanoate dehalogenase superfamily. The araL gene has been cloned, over-expressed in Escherichia coli and its product purified to homogeneity. The enzyme displays phosphatase activity, which is optimal at neutral pH (7.0) and 65 °C. Substrate screening and kinetic analysis showed AraL to have low specificity and catalytic activity towards several sugar phosphates, which are metabolic intermediates of the glycolytic and pentose phosphate pathways. On the basis of substrate specificity and gene context within the arabinose metabolic operon, a putative physiological role of AraL in the detoxification of accidental accumulation of phosphorylated metabolites has been proposed. The ability of AraL to catabolize several related secondary metabolites requires regulation at the genetic level. In the present study, using site-directed mutagenesis, we show that the production of AraL is regulated by a structure in the translation initiation region of the mRNA, which most probably blocks access to the ribosome-binding site, preventing protein synthesis. Members of haloalkanoate dehalogenase subfamily IIA and IIB are characterized by a broad-range and overlapping specificity anticipating the need for regulation at the genetic level. We provide evidence for the existence of a genetic regulatory mechanism controlling the production of AraL.

de Sanctis, D, Inácio JM, Lindley PF, de Sá-Nogueira I, Bento I.  2010.  New evidence for the role of calcium in the glycosidase reaction of GH43 arabinanases. FEBS Journal. 277:4562-4574. Abstract

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.

Ferreira, MJ, de Sá-Nogueira I.  2010.  A Multitask ATPase Serving Different ABC-Type Sugar Importers in Bacillus subtilis.. Journal of Bacteriology. 192:5312-5318., Number 20 Abstract

Bacillus 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.

Inácio, JM, Correia IL, de Sá-Nogueira I.  2008.  Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis. Microbiology. 154:2719-2729., Number 9 Abstract

Bacillus subtilis produces α-L-arabinofuranosidases (EC; 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°C, with p-nitrophenyl-α-L-arabinofuranoside as substrate, gave an apparent Km of 0.498 mM and 0.421 mM, and Vmax of 317 U mg−1 and 311 U mg−1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50°C and 60°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 α-1,5-L-arabinan and α-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.

de Sanctis, D, Bento I, Inácio JM, Custódio S, de Sá-Nogueira I, Carrondo MA.  2008.  Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-α-arabinanase from Bacillus subtilis Acta Crystallographica Section F. 64:636–638., Number 7 Abstract

Two Bacillus subtilis extracellular endo-1,5-α-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-α-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 Å, α = 82.3, β = 87.9, ɣ = 63.6°, and P212121, with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 Å. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 Å resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.

Inácio, JM, de Sá-Nogueira I.  2008.  Characterization of abn2 (yxiA), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation. Journal of Bacteriology. 190:4272-4280., Number 12 Abstract

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-α-1,5-L-arabinanase (EC 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-α-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent Km of 2.0 mg ml−1 and Vmax of 0.25 mmol min−1 mg−1 at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a σA-dependent and a σH-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.

Inácio, JM, de Sá-Nogueira I.  2007.  trans-Acting Factors and cis Elements Involved in Glucose Repression of Arabinan Degradation in Bacillus subtilis.. Journal of Bacteriology. 189:8371-8376., Number 22 Abstract

In Bacillus subtilis, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one cre each in the promoter regions of the abnA and xsa genes and to two cres in the araABDLMNPQ-abfA operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.

Franco, IS, Mota LJ, Soares CM, de Sá-Nogueira I.  2007.  Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon. Nucleic Acids Research. 35:4755-4766., Number 14 Abstract

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.

de Sá-Nogueira, I.  2007.  Regulação da Transcrição em Procariontes.. O mundo do RNA: novos desafios e perspectivas futuras. (CM Arraiano, A. Fialho, Eds.).:23-42.: Edições Técnicas Lidel
Franco, IS, Mota LJ, Soares CM, de Sá-Nogueira I.  2006.  Functional Domains of the Bacillus subtilis Transcription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding. Journal of Bacteriology. 188:3024-3036. Abstract

The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.

Leal, TF, de Sá-Nogueira I.  2004.  Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis.. FEMS Microbiology Letters. 241:41-48. Abstract

Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-α-L-arabinan.

Raposo, MP, Inácio JM, Mota LJ, de Sá-Nogueira I.  2004.  Transcriptional Regulation of Genes Encoding Arabinan-Degrading Enzymes in Bacillus subtilis. Journal of Bacteriology. 186:1287-1296., Number 5 Abstract

Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode α-L-arabinofuranosidases (EC Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-α-1,5-arabinanase (EC Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from σA-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA. The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

Inácio, JM, Costa C, de Sá-Nogueira I.  2003.  Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis. Microbiology. 149:2345-2355., Number 9 Abstract

The Bacillus subtilis proteins involved in the utilization of L-arabinose are encoded by the araABDLMNPQ–abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription ‘roadblocking’ of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.

Spencer-Martins, I, de Sá-Nogueira I.  2003.  Biotecnologia microbiana. Biotecnologia. (N. Lima, M. Mota, Eds.).:249-265.: Ediçõs Técnicas Lidel