http://hdl.handle.net/10362/3244 2013-05-24T06:03:39Z http://hdl.handle.net/10362/6165 Title: Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis Authors: Godinho, Lia M.; Sá-Nogueira, Isabel de 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. Description: FEBS journal, Volume 278, Issue 14, pages 2511-2524, July 2011 2011-01-01T00:00:00Z http://hdl.handle.net/10362/4205 Title: Functional domains of Bacillus subtilis transcription factor AraR and identification of aminoacids important for nucleoprotein complex assembly and effector-binding. Authors: Sá-Nogueira, Isabel de; Franco, Irina Saraiva; Mota, Luís Jaime; Soares, Cláudio Manuel 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. Description: Journal of Bacteriology (Apr 2006) 3024-3036 2006-03-01T00:00:00Z http://hdl.handle.net/10362/4204 Title: Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon. Authors: Sá-Nogueira, Isabel de; Franco, Irina Saraiva; Mota, Luís Jaime; Soares, Cláudio Manuel 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. Description: Nucleic Acid Research (2007) Vol.37 N. 14 4755-4766 2007-07-05T00:00:00Z http://hdl.handle.net/10362/4203 Title: trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis. Authors: Sá-Nogueira, Isabel de; Inácio, José Manuel 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. Description: Journal of Bacteriology (Nov 2007) 8371-8376 2007-11-01T00:00:00Z