AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´ -Nogueira PCR, with primers ARA444 and ARA460 (Table 1), and the resulting 1262 bp fragment was digested with BamHI and EcoRI and cloned into pMAD BamHI-EcoRI, yielding pLG10. This plasmid harboring an in-frame deletion of araL was used for integration and generation of a clean deletion in the B. subtilis chromosome, as described previ- ously by Arnaud et al. [37]. The in-frame deletion was then conﬁrmed by DNA sequencing and the resulting strain was named IQB832. Transformation of B. subtilis was per- formed as described previously by Anagnostopoulos & Spizizen [38]. Construction of an-inframe araL¢-¢lacZ fusion and integration at an ectopic site To construct plamid pLG25, the arabinose operon pro- moter region ()81 to +129, relative to the transcriptional start site) was ampliﬁed from chromosomal DNA of the B. subtilis wild-type strain 168T+ using oligonucleotides ARA28 and ARA451 (Table 1). The primers introduced unique EcoRI and HindIII restriction sites and the result- ing fragment was sub-cloned into the same sites of the cloning vector pLG1 (L. M. Godinho & I. de Sa´ Nogueira, unpublished results). Sequentially, the 5¢-end of the araL coding region comprising the rbs (position +3910 to +4020, relative to the transcriptional start site of the operon) was ampliﬁed from the wild-type strain with oligo- nucleotides ARA253 and ARA477 (Table 1), which carry unique XbaI and BamHI restriction sites and allow the insertion of this fragment between the NheI and BamHI sites of pLG1. In the resulting plasmid, a deletion of the araA rbs and araA start site present in the arabinose pro- moter region (Para) was performed by overlapping PCR using two set of primers: ARA358 and ARA514, and ARA515 and ARA516 (Table 1). The resulting fragment of 216 bp, comprising the arabinose promoter region (Para) from )81 to +80 fused to the 5¢-end of the araL coding region from +3952 to +4007, was inserted into the vector pAC5 (Table 1), yielding pLG25. Plasmid pLG25 carries a translational fusion between codon 10 of araL and codon 7 of E. coli lacZ. pLG25 was used as template for site-directed mutagenesis experiments using the mutagenic oligonucleotides set ARA509 and ARA510 (Table 1), as described above. This pair of primers gener- ated a C ﬁ A substitution at the 5¢-end of the araL cod- ing region (Figs 1 and 6A). The substitution gave rise to a mutation in the residue at position 9 (Thr to Lys) in the resulting plasmid pLG26. pLG26 was then used as tem- plate for site-directed mutagenesis using primers ARA549 and ARA550, which allowed a C ﬁ G substitution (Thr to Arg) in position 9, thus giving rise to pLG28. The oli- gonucleotide set ARA551 and ARA552 introduced a dou- ble point mutation at the 5¢-end of the araL coding sequence. Using pLG26 as template, the set of primers caused T ﬁ C (Arg to Pro) and C ﬁ G (Thr to Arg) mutations in the second and ninth positions, respectively, yielding pLG27. Plasmid pLG29 was obtained using site- directed mutagenesis from pLG26, using the oligonucleo- tide pair ARA553 and ARA554, giving rise to a G ﬁ T substitution (Arg to Leu) in position 2. The constructions were conﬁrmed by DNA sequencing. DNA from plasmids pLG25, pLG26, pLG27, pLG28 and pLG29, carrying the different araL¢-¢lacZ translational fusions, was used to transform B. subtilis strains (Table 1) and the fusions ectopically integrated into the chromosome via double recombination with the amyE gene back and front sequences. This event led to the disruption of the amyE locus and was conﬁrmed as described previously [14]. b-Galactosidase activity assays Strains of B. subtilis harboring the transcriptional lacZ fusions were grown were in liquid C minimal medium [14] supplemented with casein hydrolysate 1% (w ⁄ v), and arabi- nose was added to the cultures, when necessary, at a ﬁnal concentration of 0.4% (w ⁄ v), as described previously [14]. Samples of cell culture (100 lL) were collected 2 h (i.e. exponential growth phase) after induction and the level of accumulated b-galactosidase activity was determined by incubation for 30 min at 28 C with the chromogenic sub- strate, as described previously [14]. Acknowledgements We would like to thank Jo¨ rg Stu¨ lke for helpful discus- sions. This work was partially funded by grant no. PPCDT ⁄ BIA-MIC ⁄ 61140 ⁄ 2004 from Fundac¸ a˜o para a Cieˆ ncia e Tecnologia, POCI and FEDER to I.S.-N. 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