TABLE 2.

B. subtilis strains, plasmids, and primers used in this study

Strain, plasmid or oligonucleotide	Descriptiona	Reference, source, or site
Strains
EB6	hisA1 argC4 metC3	L5087 (6)
EB315	hisA1 argC4 metC3 amyE::xylR PxylA yacN cat86	This work
EB323	hisA1 argC4 metC3 amyE::xylR PxylA yacN cat86 yacN::spec	This work
Plasmids
pBS-yacNflank	pBluescript with yacN flanks from EB6	This work
pBS-yacNflankspec	pBluescript with Specr cassette inserted between yacN flanking sequence	This work
pSWEET	B. subtilis gene replacement plasmid described by Bhavsar and coworkers	4
pSWEET-yacN	pSWEET with wild-type yacN from EB6	This work
pUS19	pUC19 derivative containing Specr cassette	P. Levin
Oligonucleotides
yacN-Fb	5′-GGGATTAATTAAGGAGAAGGCGCTGTAAAGGGAGAAGAAACAAATGTTTAGAATTGGACAAG GATTTGATGTG-3′	PacI
yacN-R	5′-GGGAGCTAGCTTAGCCTTTTTGTATCAGTACTGTCGCCTG-3′	NheI
yacN-a	5′-GGAAGGATCCGGAGCGGGAGCACTTTCAGC-3′	BamHI
yacN-bc,d	5′-CACGCAATAACCTGCCCGGGCCAAATTTATAACCTAAACGTGTTTATTCCCACTTTCT GATTCC ATGATAGC-3′	SrfI
yacN-cc	5′-GTTATAAATTTGGCCCGGGCAGGTTATTGCGTGCTTGTTTTGATGCCGGGTCTATTTGGTGG-3′	SrfI
yacN-d	5′-GGAAGGATCCGTTACGCTCGTACTGACGGTATGG-3′	BamHI
spec-F	5′-GGTTTACACTTACTTTAGTTTTATGGAAATGAAAGATC-3′
spec-R	5′-TTATAATTTTTTTAATCTGTTATTTAAATAGTTTATAG-3′

^aUnderlined sequences indicate restriction enzyme sites.

^bBoldface sequence indicates sequence upstream of yacM including the ribosome binding site.

^cBoldface sequence indicates complementarity between yacN-b and yacN-c.

^dBoldface and italic sequence indicates a conservative histidine mutation in yacM to delete the yacN start codon.