TABLE 1.

Primers used in this study

Primer	Sequence (5′→3′)a	Amplified fragment/cloning strategy
891	GGGGTACCGGTGTAATGATTCAAAGTG	The 472-bp PCR vprA internal fragment was double digested with KpnI/XbaI and cloned into a KpnI/XbaI doubly digested pUCE191 plasmid, generating pUCE191-vprA
892	GCTCTAGATCCCGTCCTGAGCCATTTTG
965	ATGACGTTAGCAAAACATGA	Amplification with oligonucleotide 1233 to corroborate the correct insertion of pUCE191-vprA into the L. plantarum WCFS1 chromosome
1233	AGCGGATAACAATTTCACACAGGA	24-mer reverse sequencing primer (−48) for pUCE19/pUCE191
1470	CATGCCTGGAACCTGTGTTG	Amplification of a 62-bp vprA fragment for real-time quantitative PCR
1471	TGCCGACCGGAATTGC	Amplification with oligonucleotide 1690 of an 804-bp vprA-lp_3127 intergenic region
1516	TAACTTTAAGAAGGAGATATACATATGACGTTAGCAAAACATGATTCAT	The 1.5-kb vprA gene amplified with oligonucleotides 1516 and 1517 was introduced into vector pURI3-Cter by using an LICb strategy
1517	GCTATTAATGATGATGATGATGATGATGACTAACGATTGACTGCTGGTG
1633	CAGCGGTACCTATTACCGGCCTTA	The 308-bp PCR vprR internal fragment was doubly digested with KpnI/XbaI and cloned into a KpnI/XbaI doubly digested pUCE191 plasmid, generating pUCE191-vprR
1634	GTTGAGCTTTCTAGACTGTAATAT
1635	ATGGATCTTGACCGGCTACAGA	Amplification with oligonucleotide 1233 to corroborate the correct insertion of pUCE191-vprR into the L. plantarum WCFS1 chromosome
1657	GAYVTNGTNGTNGTNGG	Degenerate oligonucleotides coding for the VprA conserved motifs D(V/L/I)VVVG (1657) and GLYAAG (1658); they are used to amplify a 1.3-kb internal fragment of vprA in lactic acid bacteria
1658	CCNGCNGCRTANAGNCC
1684	CAACGGTATAGTTATCATTCGC	Amplification of a 613-bp lp_3123-vprR intergenic region
1685	TGTTAGTTGCGTTGAACCAAGC
1686	TGCTCATAGTCTTGAATAAACG	Amplification of a 596-bp vprR internal fragment
1687	GAATATCGTTCACAGCGGACAG
1688	CAGTAGGAACATTAAATTGACG	Amplification of an 800-bp vprR-vrpA intergenic region
1689	TCGCCGGCACCCATTGCTTGTACG
1690	ACTGGTTGTAGTACTGCCAGTTGC	Amplification with oligonucleotide 1470 of an 804-bp vprA-lp_3127 intergenic region
1719	GCTGCGCAACAAGGCTATG	Amplification of a 54-bp vprR fragment for real-time quantitative PCR
1720	CCGGGACCGGTTTGATTT
1810	GCCACGTTCTCATTAAGGTCCGC	Amplification of a 617-bp lp_3123 internal fragment
1811	CTTAATTGGTCGTCAACAACAGG
1812	CATTGATGTCAGCAATTGGCTAGC	Amplification of a 573-bp lp_3127 internal fragment
1813	CACTAGCTGCCATCTTAGCACCAC

^aR = G or A; Y = C or T; V = A, C or G; and N = G, A, C or T. Engineered restriction sites are underlined; nucleotides pairing the vector sequence are italicized.

^bLIC, ligation-independent cloning.