L. M. Godinho and I. de Sa´-Nogueira AraL sugar phosphatase from B. subtilis Table 1. Plasmids, oligonucleotides, and E. coli and B. subtilis strains used in the present study. Arrows indicate transformation and point from the donor DNA to the recipient strain. The restriction sites used are underlined, as are single-nucleotide point mutations. Relevant construction, genotype or sequence (5¢- to 3¢) Source or Reference Expression vector allowing N- or C-terminal His6 tag insertion; T7 promoter, kan Plasmid used for allelic replacement in Gram-positive bacteria, bla, erm Plasmid used for generation of lacZ translational fusions and integration at the B. subtilis amyE locus, bla, cat araL sequence with the ﬁrst putative araL start codon cloned in the pET30a vector pMAD derivative with an in frame deletion DaraL araL sequence with mutated GTG codon (valine at position 8) to ATG (methionine) cloned in the pET30a vector araL sequence with the putative second araL start codon cloned in the pET30a vector A pLG12 derivative with a mutation in the araL sequence GGC to GAC (Gly12 to Asp) A pAC5 derivative that contains a translational fusion of araL to the lacZ gene under the control of the arabinose operon promoter (Para) (recA1 endA1 gyrA96 thi-1 hsdr17 supE44 relA1 lac [F’ proAB lacIq ZDM15 Tn10 (Tetr)] fhuA2 D(argF-lacZ)U169 phoA glnV44 F80 D(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 F) ompT hsdSB(rB) mB)) gal dcm (DE3) pLysS (CmR) A pLG25 derivative with a mutation in the araL sequence ACG to AAG (Thr9 to Lys) Present study Plasmid, strain or oligonucleotide Plasmids pET30a pMAD pAC5 pLG5 pLG10 pLG11 pLG12 pLG13 pLG25 pLG26 E. coli strains XL1 blue DH5a BL21(DE3)pLysS B. subtilis strains 168T+ IQB832 IQB215 IQB847 IQB848 IQB849 IQB851 IQB853 IQB855 IQB857 Oligonucleotides ARA28 ARA253 ARA358 ARA439 ARA440 ARA444 ARA451 ARA456 ARA457 ARA458 ARA459 ARA460 ARA477 ARA486 ARA487 ARA509 ARA510 ARA514 ARA515 Novagen [37] [39] Present study Present study Present study Present study Present study Present study Stratagene Gibco-BRL [40] [12] pLG10 ﬁ 168T+ [14] pLG25 ﬁ 168T+ pLG25 ﬁ IQB215 pLG26 ﬁ 168T+ pAC5 ﬁ 168T+ pLG27 ﬁ 168T+ pLG28 ﬁ 168T+ pLG29 ﬁ 168T+ Prototroph DaraL DaraR::km amyE::Para-araL’-’lacZ cat DaraR::km amyE::Para-araL’-’lacZ cat amyE::Para-araL’ (C ﬁ A) -’lacZ cat amyE:: ‘lacZ cat amyE::Para-araL’ (T ﬁ C and C ﬁ G) -’lacZ cat amyE::Para-araL’ (C ﬁ G) -’lacZ cat amyE::Para-araL’ (C ﬁ A and G ﬁ T) -’lacZ cat CCTATTGAATTCAAAAGCCGG TAACCCCAATCTAGACAGTCC CTGCTGTAATAATGGGTAGAAGG GGAATTCCATATGCGTATTATGGCCAG TATTTACTCGAGAATCCCCTCCTCAGC CGGGATCCACCGTGAAAAAGAAAGAATTGTC GAATTCATAAAGAAGCTTTGTCTGAAGC CGGCGCGTCATATGGCCAGTCATGATA TGATACGCATATGTCACCGGCTGGC CTCAGCCAATTTGGTTACATCCTTGTCCAAGTCAATCAGAATGCCAGCCGGTGCCAC GTGTCACCGGCTGGCATTCTGATTGACTTGGACAAGGATGTAACCAAATTGGCTGAG CGTGAATTCACCGAGCATGTCACCAAAGCC AATCAGAATGGGATCCGGTGA CGGCTGACATTCTGATTGACTTGGACGG CAATCAGAATGTCAGCCGGTGACACAGG CC AGT CAT GAT AAG CCT GTG TCA CCG CGG TGA CAC AGG CTT ATC ATG ACT GG TAATACGCATTTGCTC CGT GTT TTC GTC ATA AAA TAA AAC GCT TTC AAA TAC GTATTTGAAAGCGTTTTATTTTATGACGAA AAC ACG GAG CAA ATG CGT ATT A FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2515