L. M. Godinho and I. de Sa´ -Nogueira AraL sugar phosphatase from B. subtilis A B Fig. 6. Regulation of araL in B. subtilis. (A) Site-directed mutagenesis at the 5¢-end of the araL gene. The secondary structure of the ara- ABDLMNPQ-abfA mRNA at the 5¢-end of the araL coding region is depicted. An arrow highlights the mutated nucleotide (circled) located at the beginning of the araL coding region. The ribosome-binding site, rbs, is boxed. (B) Expression from the wild-type and mutant araL¢-¢lacZ translational fusions. The B. subtilis strains IQB847 (Para-araL¢-lacZ), IQB849 [Para-araL¢ (C ﬁ A)-¢lacZ], IQB857 [Para-araL¢ (C ﬁ A and G ﬁ T)-¢lacZ], IQB855 [Para-araL¢ (C ﬁ G)-¢lacZ] and IQB853 [Para-araL¢ (C ﬁ G and G ﬁ C)-¢lacZ] were grown on C minimal medium supple- mented with casein hydrolysate in the absence (non-induced) or presence (induced) of arabinose. Samples were analyzed 2 h after induction. The levels of accumulated b-galactosidase activity represent the mean ± SD of three independent experiments, each performed in triplicate. A schematic representation of the translation fusion is depicted and the point mutations in the stem-loop structure are indicated by an aster- isk. The free energy of the wild-type (WT) and mutated secondary structures, calculated by DNASIS, version 3.7 (Hitachi Software Engineering Co. Ltd), are shown. clear boundaries deﬁning physiological substrates, reg- ulation at the genetic level was anticipated [13]. In the present study, we show for the ﬁrst time that a genetic regulatory mechanism controls the expression ⁄ produc- tion of a member of the NagD family, AraL. The AraL enzyme encoded by the arabinose meta- bolic operon araABDLMNPQ-abfA was previously shown to be dispensable for arabinose utilization in a strain bearing a large deletion comprising all genes downstream from araD. However, this strain displayed some growth defects [12]. To conﬁrm this hypothesis, an in-frame deletion mutation in the araL gene was generated by allelic replacement, aiming to minimize the polar effect on the genes of the araABDLMNPQ- abfA operon located downstream of araL (Fig. 1). The physiological effect of this knockout mutation in B. subtilis (strain IQB832 DaraL; Table 1) was assessed by determining the growth kinetics parameters using glucose and arabinose as the sole carbon and energy source. In the presence of glucose and arabinose, the doubling time of the mutant (49.7 ± 0.3 and 52.4 ± 0.1 min, respectively) is comparable to that of the wild-type strain (46.6 ± 0.4 and 52.2 ± 0.5 min, respectively), indicating both the stability of the strain bearing the in-frame deletion and the fact that the AraL enzyme is not involved in l-arabinose utilization. The substrate speciﬁcity of AraL points to a biological function within the context of carbohydrate metabo- lism. The location of the araL gene in the arabinose metabolic operon, together with the observation that AraL is active towards d-xylulose 5-phosphate, a metabolite resulting from l-arabinose catabolism, sug- gests that AraL, similar to other HAD phosphatase members, may help the cell to get rid of phosphory- lated metabolites that could accumulate accidentally via stalled pathways. The arabinose operon is under the negative control of the transcription factor AraR and, in an araR-null mutant, the expression of the operon is constitutive. In a previous study [14], the addition of arabinose to an early-exponentially grow- ing culture of this mutant resulted in immediate cessa- tion of growth. It was speculated that this effect could be the result of an increased intracellular level of arabi- nose, which would consequently cause an increase in the concentration of the metabolic sugar phosphate intermediates that are toxic to the cell [14]. Thus, we may hypothesize that AraL possibly plays a role in the dephosphorylation of substrates related to l-arabinose metabolism, namely l-ribulose phosphate and ⁄ or d-xylulose phosphate. In addition, because of its FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2519