Strain constructions
Table S2 lists all plasmids and bacterial strains used in these studies. To construct
strains that produce a c-Myc-tagged YwbN variant, the ywbN gene was amplified by
PCR with primers JJ65ywm and JJ66ywm. Primer JJ65ywm (5'-GCT CTA GAA
TGT GCT ATA AAA GGA G-3') contains an XbaI site and upstream sequences of
ywbN. Primer JJ66ywm (5'-GCG GAT CCT TAG TTC AAA TCT TCC TCA CTG
ATC AAT TTC TGT TCT GAT TCC AGC AAA CGC TGG GC-3') contains a
BamHI site, the sequence specifying the human c-Myc epitope (indicated in bold; Evan
et al., 1985), and the 3’ sequences of ywbN. The resulting PCR product, which contains
the ywbN-myc gene, was cleaved with XbaI and BamHI and ligated into the SpeI and
BamHI sites of pX (Kim et al., 1996). This resulted in plasmid pXYNm1, which carries
the ywbN-myc gene under the transcriptional control of the xylose-inducible xylA
promoter. The xylose-inducible YwbN-myc producing strain 168 X-ywbN was obtained
by a double cross-over recombination event that resulted in the introduction of the xylAywbN-myc cassette of pXYNm1 into the chromosomally-located amyE gene of B.
subtilis 168.
To construct B. subtilis tatAc1 and tatAc2, the tatAc region was amplified by PCR
with primer JJ45Acd (5’-GGA ATT CAG AAA GTC TGG GAG-3’) containing an
EcoRI site and 5’ sequences of tatAc, and primer JJ46Acd (5’-GCT CTA GAA ATA
TAC ATA TAG TGC-3’) containing an XbaI site and 3’ sequences of tatAc. Next, the
PCR-amplified fragment was cleaved with EcoRI and XbaI and ligated into the EcoRI
and XbaI sites of pUC21 or pUK21 (Vieira & Messing, 1991), resulting in pJAc1 and
pJKAc1, respectively. Plasmid pJAc2 was obtained by ligating a pKM1-derived Km
resistance marker, flanked by HindIII restriction sites, into the unique HindIII site of the
tatAc gene in pJAc1. Plasmid pJKAc6 was obtained by replacing an internal HindIIINaeI fragment of the tatAc gene in pJKAc1 with a pDG1515-derived Tc resistance
marker (Guérot-Fleury et al. 1995), flanked by HindIII and SmaI restriction sites. Finally,
B. subtilis tatAc1 and B. subtilis tatAc2 were obtained by a double cross-over
recombination between the chromosomal tatAc region and the disrupted tatAc genes of
pJAc2 or pJKAc6, respectively. For a better understanding of the construction of tatA
mutants, this is schematically represented in Fig. S2.
To construct B. subtilis tatAd, two DNA fragments containing 5' or 3’ sequences of
tatAd were amplified by PCR. The 5’ fragment of tatAd was amplified with primer
JJ47Add (5’-GGA ATT CCC AGA TAT GCA GCT GTA CC-3’), which contains an
EcoRI site and sequences upstream of tatAd, and primer JJ47Ad2 (5’-CCC AAG CTT
GAG AAT CAA GCC CGG TAT TCC-3’), which contains a HindIII site and codons
16-37 of the tatAd gene. The 3’ fragment of tatAd was amplified with primer JJ48Ad2
(5’-CCC AAG CTT GAC AGC GGT AAA GCA GG-3’), which contains a HindIII site
and codons 179-196 of the tatAd gene, and primer JJ48Add (5’-CGG GAT CCG GAG
GAT AAA TGA GTC-3’), which contains a BamHI site and sequences downstream of
tatAd. Next, these PCR-amplified fragments were cleaved with EcoRI/HindIII and
HindIII/BamHI respectively, and ligated into the EcoRI and BamHI sites of pUC21. This
resulted in pJAd1. Plasmid pJAd2 was obtained by ligating a pKM1-derived Km
resistance marker, flanked by HindIII restriction sites, into the unique HindIII site of the
tatAd region in pJAd1. Finally, B. subtilis tatAd was obtained by a double cross-over
recombination between the disrupted tatAd region of pJAd2 and the chromosomal tatAd
region.
To construct B. subtilis tatAy, two DNA fragments containing 5' or 3’ sequences of
tatAy were amplified by PCR. The 5’ fragment of tatAy was amplified with primer
JJ49Ayd (5’-GGG GTA CCT TAA AGA ATC TGC ATG CG-3’), which contains a
KpnI site and sequences upstream of tatAy, and primer JJ49Ay2 (5’-TGC GGT CGA
CAG CAA GGC TTC CAG GAC CG-3’), which contains a SalI site and codons 9-29
of the tatAy gene. The 3’ fragment of tatAy was amplified with primer JJ50Ay2 (5’-.ACG
CGT CGA CTA AAG GAT TAA CGA GTG ATG-3’), which contains a SalI site and
codons 126-145 of the tatAy gene, and primer JJ50Ayd (5’-CGG GAT CCC AGA
AGA CAC GTC CCG-3’), which contains a BamHI site and sequences downstream of
tatAy. Next, these PCR-amplified fragments were cleaved with KpnI/SalI and
SalI/BamHI respectively, and ligated into the KpnI and BamHI sites of pUC21. This
resulted in pJAy1. Plasmid pJAy4 was obtained by ligating a pUC19E-derived Em
resistance marker, flanked by SalI restriction sites, into the unique SalI site of the tatAy
region in pJAy1. Finally, B. subtilis tatAy was obtained by a double cross-over
recombination between the disrupted tatAy region of pJAy4 and the chromosomal tatAy
region.
The construction of B. subtilis tatAdCd was performed as described for B. subtilis
tatAdCd (Jongbloed et al., 2002), except for the use of a pKM1-derived Km resistance
marker, flanked by AccI restriction sites, for the replacement of the tatAd-tatCd region.
To construct multiple tat mutants, single, double and triple mutants were transformed
with chromosomal DNA or linearized plasmid DNA containing a disrupted tat gene or
operon. The absence of intact tat genes from tat mutants was verified by PCR.
Finally, tat or sip X-ywbN mutants were obtained by transformation of the respective tat
or sip mutants with plasmid pXYNm1, resulting in the integration of the xylA-ywbN-myc
cassette into their chromosomes.
To construct plasmid pCAy1, the tatAy gene was amplified by PCR with the
primers JW01Ay2 (5’-ACG CGT CGA CGA ATA GAG GGA AAG GAG G-3’)
containing a SalI site, and JW02Ay2 (5’-GGA ATT CAT TCG TCA TCC TAA C-3’)
containing an EcoRI site. The amplified fragment was cleaved with SalI and EcoRI,
and cloned in the corresponding sites of pGDL48 (Tjalsma et al., 1998), resulting in
pCAy1. To construct plasmid pCCy1, the tatCy gene was amplified by PCR with the
primers JW03Cy2 (5’-ACG CGT CGACGA GTG ATG AAG AGG-3’) containing a
SalI site, and JW04Cy2 (5’-GGA ATT CTC GCA TGA GAA ATG G-3’) containing
an EcoRI site. The amplified fragment was cleaved with SalI and EcoRI, and cloned
in the corresponding sites of pGDL48, resulting in pCCy1. To construct plasmid
pCACy1, the tatAy-tatCy region was amplified by PCR with the primers JW01Ay2 (5’ACG CGT CGA CGA ATA GAG GGA AAG GAG G-3’) containing a SalI site,
and JW04Cy2 (5’-GGA ATT CTC GCA TGA GAA ATG G-3’) containing an EcoRI
site. The amplified fragment was cleaved with SalI and EcoRI, and cloned in the
corresponding sites of pGDL48, resulting in pCACy1.