AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´-Nogueira capacity to catabolize other related secondary metabo- lites, this enzyme needs to be regulated. Moreover, the araL gene is under the control of the operon promoter, which is a very strong promoter, and basal expression in the absence of inducer is always present [14]. The second level of regulation within the operon that oper- ates in araL expression will act to drastically reduce the production of AraL. Experimental procedures Substrates pNPP was purchased from Apollo Scientiﬁc Ltd (Stockport, UK) and d-xylulose 5-phosphate, glucose 6-phosphate, fruc- tose 6-phosphate, fructose 1,6-bisphosphate, ribose 5-phos- phate, d-arabinose 5-phosphate, galactose 1-phosphate, glycerol 3-phosphate, pyridoxal 5-phosphate, thiamine monophosphate, ATP, ADP and AMP were obtained from Sigma-Aldrich (St Louis, MO, USA). Bacterial strains and growth conditions E. coli strains XL1Blue (Stratagene, La Jolla, CA, USA) or DH5a (Gibco-BRL, Carlsbad, CA, USA) were used for molecular cloning work and E. coli BL21 (DE3)(pLysS) was used for the overproduction of AraL (Table 1). E. coli strains were grown in LB medium [33] or in auto-induction medium [20]. Ampicillin (100 lgÆmL)1), chloramphenicol (25 lgÆmL)1), kanamycin (30 lgÆmL)1), tetracycline (12 lgÆmL)1) and IPTG (1 mm) were added as appropriate. B. subtilis was grown in liquid LB medium, LB medium solidiﬁed with 1.6% (w ⁄ v) agar, with chloramphenicol (5 lgÆmL)1), erythromycin (1 lgÆmL)1) and X-Gal (50 lgÆmL)1) being added as appropriate. Growth kinetics parameters of the wild-type and mutant B. subtilis strains were determined in CSK liquid minimal medium [34], as described previously [27]. Cultures were grown on an Aqua- tron Waterbath rotary shaker (Infors HT, Bottmingen, Switzerland), at 37 C (unless stated otherwise) and 180 r.p.m., and A600 was measured in an Ultrospec 2100 pro UV ⁄ Visible Spectrophotometer (GE Healthcare Life Sciences, Uppsala, Sweden). DNA manipulation and sequencing DNA manipulations were carried out as described previ- ously by Sambrook et al. [35]. Restriction enzymes were purchased from MBI Fermentas (Vilnius, Lithuania) or New England Biolabs (Hitchin, UK) and used in accor- dance with the manufacturer’s instructions. DNA ligations were performed using T4 DNA Ligase (MBI Fermentas). DNA was eluted from agarose gels with GFX Gel Band Puriﬁcation kit (GE Healthcare Life Sciences) and plasmids were puriﬁed using the Qiagen Plasmid Midi kit (Qiagen, Hilden, Germany) or QIAprep Spin Miniprep kit (Qia- gen). DNA sequencing was performed with ABI PRIS Big- Dye Terminator Ready Reaction Cycle Sequencing kit (Applied Biosystems, Carlsbad, CA, USA). PCR ampliﬁca- tions were conducted using high-ﬁdelity Phusion DNA polymerase from Finnzymes (Espoo, Finland). Plasmid constructions Plasmids pLG5, pLG11 and pLG12 are pET30a derivatives (Table 1), which harbor different versions of araL bearing a C-terminal His6-tag, under the control of a T7 inducible promoter. The coding sequence of araL was ampliﬁed by PCR using chromosomal DNA of the wild-type strain B. subtilis 168T+ as template and different sets of primers. To construct pLG5, oligonucleotides ARA439 and ARA440 (Table 1) were used and introduced unique NdeI and XhoI restriction sites at the 5¢ and 3¢ end, respectively, and the resulting PCR product was inserted into pET30a digested with the same restriction enzymes. Using the same procedure, primers ARA457 and ARA440 (Table 1) gener- ated pLG11. ARA457 introduced a mutation, which substi- tutes Val at position 8 to Met (Fig. 1). Plasmid pLG12 was constructed with primers ARA456 and ARA440. Primer ARA456 inserted an NdeI restriction site in the araL sequence at the second putative start codon (Fig. 1). Site-directed mutagenesis Vector pLG12 was used as template for site-directed muta- genesis experiments using the mutagenic oligonucleotides set ARA486 and ARA487 (Table 1). This pair of primers generated a G ﬁ A substitution at the 5¢-end of the araL coding region (Fig. 1). This substitution gave rise to a mutation in the residue at position 12 (Gly to Asp) in the resulting plasmid pLG13. PCR was carried out using 1 · Phusion GC Buffer (Finnzymes), 0.2 lm primers, 200 lm dNTPs, 3% dimethylsulfoxide, 0.4 ngÆlL)1 pLG12 DNA and 0.02 UÆlL)1 of Phusion DNA polymerase in a total volume of 50 lL. The PCR product was digested with 10 U of DpnI, at 37 C, overnight. The mutation was conﬁrmed by sequencing. Overproduction and puriﬁcation of recombinant AraL proteins in E. coli Small-scale growth of E. coli BL21(DE3) pLysS cells har- boring pLG5, pLG11, pLG12 and pLG13 was performed to assess the overproduction and solubility of the recombi- nant proteins. Cells were grown at 37 C, at 180 r.p.m. and 1 mm IPTG was added when A600 of 0.6 was reached. Cul- tures were then grown for an additional 3 h at 37 C and 180 r.p.m. Whenever protein solubility was not observed, 2520 FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS