SUPPLEMENTAL MATERIAL
Construction of strains
(1) Mutant strains carrying various deletions
Gene disruptions were constructed via homologous recombination using
PCR-generated fragments. The primary PCR-generated fragments
contained around 500 to 1,000 bp of the upstream (using primers -11 and
-12) and downstream (using primers -23 and -24) sequences of the target
gene, which overlap either end of the PCR-generated fragment using
primers -For and –Rev and containing an antibiotic gene marker. We used
the recombinant PCR method (Wach, 1996), to generate the secondary
PCR products with 3 primary PCR fragments. All constructs were verified
by genomic PCR.
(2) plsX-inducible strain
In the plsX-inducible strain NBS1014 [plsX::pMT3plsX (Pspac-plsX erm )
fabD::pfabD15 (PrepU-neo-fabD-fabG )], plsX was placed under the
control of the Pspac-IPTG inducible promoter. Construct details were
described previously (Hara et al, 2008).
(3) Strains expressing GFP- and HA-fusion proteins
Strain NBS402 [trpC2 PftsAZ-ftsA-gfp cat]
The ftsA region including its promoter was PCR-amplified from wild-type
genomic DNA using primers ftsA5′-EcoRI and ftsA3′-BamHI and cloned
between the EcoRI and BamHI sites of pGFP7C (Kuwana et al, 2006),
creating pFTSA8G. This plasmid was used to transform strain 168 for cat
resistance to generate strain NBS402, in which the gfp-fused ftsA is
expressed from the native PftsAZ promoter at the native locus on the
chromosome.
Strain NBS800 [amyE::spc(Pxyl-gfp-plsX)]
The plsX coding region was PCR-amplified from the wild-type genomic
DNA using primers plsXXhoI and plsXecoRI and cloned between the XhoI
and EcoRI sites of pSG1729 (Lewis & Marston, 1999), creating pGFP-PlsX.
The linker sequence coding a 12-amino acid (a.a.) peptide
LELPGPELPGPE was used to connect gfpmut1 to plsX. This plasmid was
used to transform strain 168 for spc resistance to generate strain NBS800.
Strain NBS1876 [amyE::spc(Pxyl-gfpA206K-plsX)]
The A206K mutation, which prevents dimerization of GFP (Landgraf et al,
2012; Zacharias et al, 2002), was introduced into the gfp part of plasmid
pGFP-PlsX using KOD-Plus Mutagenesis Kit (TOYOBO, Japan) with
gfpmut1A206K for/ gfpmut1A206K rev as the mutagenic primers, creating
pGFPA206K-PlsX. This plasmid was used to transform strain 168 for spc
resistance to generate strain NBS1876.
Strain NBS1877 [thrC::(Phy-gfp-plsX ermR)]
The gfp-plsX fragment was PCR-amplified from pGFP-PlsX using primers
PhygfpplsX(NheI) and PhyplsXR(NheI) and cloned between the NheI sites
of pHT003, creating pHT308. This plasmid was used to transform strain
168 for erm resistance to generate strain NBS1877.
Strain NBS1009 [plsX::pMHAcPlsX(plsX-HA erm –Pspac-fabD-fabG)]
The gene fragment corresponding to the C-terminal region (from the 163rd
to the carboxyl-terminal 333rd amino acid residue) of PlsX was
PCR-amplified from the wild-type genomic DNA using primers
plsX5′-BamHI and plsX3′-EcoRI and cloned between the BamHI and
EcoRI sites of pMHAc (K. Asai, unpublished data), creating pMHAcPlsX.
This plasmid was used to transform strain 168 for erm resistance and IPTG
inducibility to generate strain NBS1009 in which the HA-fused plsX is
expressed from the native promoter PfapR at the native locus on the
chromosome. Downstream genes are under the control of the
IPTG-inducible Pspac promoter.
Strain NBS1878 [spc-cfp(Bs)-ftsA]
This strain was constructed by homologous recombination using
PCR-generated fragments, which were made using primers ftsA up For and
ftsA up Rev for the downstream fragment of the ftsA gene, ftsA-spc For
and ftsA-spc Rev for the coding region of the antibiotic gene marker, and
cfp For and cfp Rev for cfp (Bs) fragment of the plsX gene, ftsA orf For and
ftsA orf Rev for the coding region of ftsA gene, as described above. These
recombinant fragments were used to transform strain 168 via homologous
recombination. Spectinomycin-resistant transformants were selected at
37°C. The linker sequence coding a 13-a.a. peptide GSAGSAGSAAGSG
was used to connect cfp to ftsA. The cyan fluorescent protein (CFP) gene,
using codons optimized for B. subtilis, was PCR amplified from plasmid
pDR200 (a gift from M. Fujita).
(4) Strain plsX-his12
NBS1517 [plsX-his12 spc] was constructed via homologous recombination
using PCR-generated fragments, which were made using primers
plsX-mutFor and plsX-His12 ORF rev for the histidine-tagged plsX
fragment, plsX-His12 spc For and plsX-spc Rev for the coding region of
the antibiotic gene marker, and plsX-23 and plsX-24 for the downstream
fragment of the plsX gene, as described above. These recombinant
fragments were used to transform strain 168 via homologous
recombination. Spectinomycin-resistant transformants were selected at
37°C.
(5) Overexpression strain
To generate vector pHT001, the PCR product containing the
chloramphenicol-resistance marker was amplified from plasmid pC194
using the primer pair catF/catR, digested with EcoRI and DraIII, and
ligated to the EcoRI and DraIII sites of pDR111 which carries a
erythromycin-resistance marker, a polylinker downstream of the Phy-spank
promoter, and the gene for the LacI repressor, between two arms of the
amyE gene (Ben-Yehuda et al, 2003). To generate vector pHT002, the PCR
product containing the erythromycin-resistance marker was amplified from
plasmid pMUTIN4 using the primer pair ermF/ermR, digested with EcoRI
and DraIII, and ligated to the EcoRI and DraIII sites of pDR111. To
generate vector pHT003, the fragment containing the Phy-spank promoter
and the gene for the LacI repressor was excised from plasmid pDR111
using the restriction enzymes EcoRI and BamHI and was ligated to the
EcoRI and BamHI sites of plasmid pDG1664 which carries an
erythromycin resistance marker and a polylinker downstream between two
arms of the thrC gene (Guerout-Fleury et al, 1996).
Strain NBS1569 [thrC::(Phy-ftsA ermR)]
The ftsA-coding region was PCR-amplified from the wild-type genomic
DNA using primers PhyftsAF(SalI) and PhyftsAR(NheI) and cloned
between the SalI and NheI sites of pHT002, creating pHT206. The
fragment including ftsA gene was excised from pHT206 using the
restriction enzyme EcoRI and BamHI and was ligated to the EcoRI and
BamHI sites of plasmid pDG1664, creating pHT402. This plasmid was
used to transform strain 168 for erm resistance to generate strain NBS1569.
Strain NBS1571 [thrC::(Phy-mciZ ermR)]
The mciZ-coding region was PCR-amplified from the wild-type genomic
DNA using primers PhymicZF(HindIII) and PhymicZR(NheI) and cloned
between the HindIII and NheI sites of pHT003, creating pHT307. This
plasmid was used to transform strain 168 for erm resistance to generate
strain NBS1571.
Strain NBS1572 [thrC::(Phy-sirA ermR)]
The sirA-coding region was PCR-amplified from the wild-type genomic
DNA using primers PhysirAF(SalI) and PhysirAR(NheI) and cloned
between the SalI and NheI sites of pHT002, creating pHT215. The
fragment including sirA gene was excised from pHT215 using the
restriction enzyme EcoRI and BamHI and was ligated to the EcoRI and
BamHI sites of plasmid pDG1664, creating pHT408. This plasmid was
used to transform strain 168 for erm resistance to generate strain NBS1572.
Screening of the plsX ts mutant strain by random PCR mutagenesis
Since plsX gene is located in the fap operon, we placed a marker gene, spc,
which did not contain its own promoter to avoid polar effects, downstream
of plsX gene. This strain, named NBS1327, showed normal growth and
morphology at high temperature (Fig. 6C) and polar effects were not
observed. Temperature-sensitive mutants of plsX were constructed via
homologous recombination of PCR-generated fragments. The primary
PCR-generated fragments contained 1) around 500 to 1,000 bp of the
upstream sequence of the plsX gene (using primers plsX-11 and plsX-12),
2) the PCR-mutagenized fragment of the plsX coding sequence using Taq
polymerase and primers plsX-mutFor and plsX-mutRev, 3) the coding
region of the antibiotic gene marker using primers plsX-spcFor and
plsX-spcRev, and 4) the downstream sequence of the plsX gene (using
primers plsX-23 and plsX-24). The mutated plsX fragment overlapped with
the 5′ end of the antibiotic gene marker and the 3′ end of the upstream
fragment of the plsX gene, while the downstream sequence of the plsX gene
overlapped with the 3′ end of the antibiotic gene marker. We used
recombinant PCR to generate the secondary PCR products with these 4
primary PCR fragments. The recombinant fragment was used to transform
strain 168 via homologous recombination. Spectinomycin-resistant
transformants were selected at 30°C and screened for the inability to grow
above 45°C to isolate plsX ts mutants.
SUPPLEMENTAL FIGURE LEGENDS
Fig. S1. Specific interaction between cell division proteins and
phospholipid synthases.
Matrices of Y2H interactions occurring between cell division proteins and
phospholipid synthases are shown. Already known interactions among
proteins related to cell division are indicated by white squares. The
interactions between PlsX and cell division proteins are indicated by red
squares. The indicated proteins are expressed as baits (BD, Gal4 BD
fusion) and/or as preys (AD, Gal4 AD fusion). Interactions between
proteins were detected on selection medium (SC-LWH) supplemented with
1 mM 3-aminotriazol after 7 days incubation at 30°C.
Fig. S2. Isolation and analysis of the PlsX complex.
Separation and visualization of protein complexes purified from cultures
containing his-tagged PlsX (strain NBS1517) by SDS-PAGE and colloidal
Coomassie staining (see Experimental Procedures). Each protein was
identified by mass spectrometry analysis.
Fig. S3. Subcellular localization of GFPA206K-PlsX in the strain
NBS1876.
Images obtained from exponentially growing cells in LB medium
containing 0.5% xylose at 37°C are shown as in Fig 1D. That is, from left
to right, FM4-64-stained membranes, GFP-PlsX, DAPI-stained DNA, and
superposition of GFP-PlsX (green), FM-4-64-stained membranes (red), and
DAPI-stained DNA (blue), respectively. Arrows with each number
corresponds to positions with the same number in the fluorescence intensity
profiles. Fluorescence intensity profiles of FM4-64 (red), GFP–PlsX
(green), and DAPI (blue) in arbitrary unit along the cell length are shown
on the bottom. Scale bar indicates 5 μm.
Fig. S4. Immunofluorecent localization of PlsX-HA.
A. Growth profiles of strain NBS1009 in LB medium with 1 mM IPTG at
37°C. Growth was monitored by OD measurements.
B. Cellular amount of PlsX-HA detected by immunoblot analysis. At each
time point, culture was collected by centrifugation. Protein samples were
subjected to SDS-PAGE followed by immunoblot analysis with anti-HA
antibodies. The cellular amount of SigA as a control was also analysed with
anti-SigA antibodies.
C. Localization of PlsX-HA was detected with a monoclonal antibody
against HA. NBS1009 strain was cultured in LB medium with 1mM IPTG
at 37°C and cells were collected from exponentially growing cultures.
Panels show from left to right, immunofluorescence of PlsX-HA,
DAPI-stained DNA, and phase-contrast images. Scale bar: 5 μm.
D. Cellular localization of GFP-PlsX before (left) and after (middle and
right) fixation procedure. In our fixation procedure (middle), culture was
fixed in growth medium with a final concentration of 2.6%
paraformaldehyde and 30 mM sodium phosphate buffer (pH 7.4) for 30
min at room temperature. In the previous reported procedure (right), culture
was fixed in growth medium with a final concentration of 2.6%
paraformaldehyde, 0.006% glutaraldehyde, and 30 mM sodium phosphate
buffer (pH 7.4) for 15 min at room temperature and 30 min on ice. Scale
bar indicates 5 μm.
Fig. S5. Effect of mciZ induction on the localization of GFP-PlsX.
A. Images of cells (strain NBS1583) before and after MciZ induction. Time
(in hour) after the addition of IPTG is indicated. MciZ was induced with
1.0 mM IPTG. Images and fluorescence intensity profiles are shown as in
Fig. S3. Scale bar indicates 5 μm.
B. Immunoblot analysis showing that GFP-PlsX remains intact after MciZ
induction. GFP-PlsX was analysed using anti-GFP antibodies. The asterisk
indicates the predicted size of free GFP. SigA, which was detected by
anti-SigA antibody, was used as a control for loading.
Fig. S6. Comparison of the localization pattern of GFP-PlsX in the rich
(LB) medium and the minimal (S7 minimal salt) medium.
A and B. The localization of GFP-PlsX in the wild-type strain (NBS1877)
and the minCD mutant (NBS1878) under growth in LB medium (A) and S7
minimal salt medium (B). GFP-PlsX was induced with 0.2 mM IPTG.
Images and fluorescence intensity profiles are shown as in Fig. S3. Scale
bar indicates 5 μm.
C. Effect of the medium condition on the cell length of the wild-type strain
and minCD mutant. Histograms show the distribution of length under the
indicated conditions. About 100 cells were measured for each strain.
D. Immunoblot analysis of GFP-PlsX in strains NBS1877, NBS1878, and
wild-type 168 under the indicated conditions. GFP-PlsX was analysed
using anti-PlsX antibodies. SigA was used as a control for loading.
Fig. S7. Effect of mciZ overexpression on the localization of GFP-PlsX in
the minCD mutant.
A. Images of strain NBS1875 cells and fluorescence intensity profiles
shown as in Fig. S3 before and after FtsA induction. Time (in hour) after
the addition of IPTG is indicated. MciZ was induced with 1.0 mM IPTG.
Scale bar indicates 5 μm.
B. Immunoblot analysis showing that GFP-PlsX remains intact after MicZ
induction. GFP-PlsX was analysed using anti-GFP antibodies. The asterisk
indicates the predicted size of free GFP. SigA was used as a control for
loading.
Fig. S8. Effect of ftsA overexpression on septum formation.
A. Colony formation of NBS1569. Strain NBS1569 was streaked on LB
plates with or without 0.2 mM IPTG and cultured overnight at 37°C.
B. Effect of ftsA overexpression on cell division. Images of strain NBS1569
cells are shown. From left to right, FM-4-64-stained membranes,
DAPI-stained DNA, superposition of FM-4-64-stained membranes (red)
and DAPI-stained DNA (blue), and phase contrast microscopy (PC, only in
the presence of IPTG), respectively, before and after FtsA induction. Time
(in hour) after the addition of IPTG is indicated. FtsA was induced with 0.2
mM IPTG. Scale bar indicates 5 µm. White arrows and the yellow arrow
indicate minicell and twisted septum respectively.
C. Immunoblot analysis of FtsA in the wild-type 168 and NBS1569 strains.
FtsA was analysed using anti-FtsA antibody. SigA was used as a control for
loading (indicated by arrow). The asterisk indicates the band of FtsA due to
using the same secondary antibody (rabbit).
Fig. S9. Effect of 3-MBA on PlsX localization.
Colocalization of GFP-PlsX and CFP–FtsA after growth for 1 h in the
presence of 3-MBA (10 mM). Images are shown as in Fig 1E. From left to
right, FM4-64-stained membranes, GFP-PlsX, superposition of GFP-PlsX
(green), FM-4-64-stained membranes (red), and CFP-FtsA (blue), and
CFP-FtsA, respectively. The arrows with each number correspond to
positions with the same number in the fluorescence intensity profiles.
Fluorescence intensity profiles of FM4-64 (red), GFP-PlsX (Green), and
CFP-FtsA (light blue) are shown on the bottom. Scale bar indicates 5 μm.
Fig. S10. Localization of GFP-FtsA in cells with DNA replication initiation
defect.
A. Images of strain NBS1584 cells before and after SirA induction. Time
(in hour) after the addition of IPTG is indicated. SirA was induced with 1.0
mM IPTG. Images and fluorescence intensity profiles are shown as in Fig.
S3.
B. Immunoblot analysis showing that GFP-FtsA remains intact after SirA
induction. GFP-FtsA was analysed using anti-GFP antibodies. The asterisk
indicates the predicted size of free GFP. SigA was used as a control for
loading.
Fig. S11. Growth of plsX mutants at several temperatures.
Colony formation of wt168, NBS1327 (plsX-spc), NBS1328 (plsXD59G),
NBS1329 (plsXL104S) and NBS1010 (plsX103) is shown. Each strain was
streaked on LB plate and grown overnight at 30°C, 39°C, or 45°C.
Fig. S12. Aberrant septum formation caused by depletion of PlsX.
A. Growth profiles of plsX-inducible strain NBS1014. NBS1014 strain was
streaked on LB pates containing 0.3 mM IPTG and incubated at room
temperature overnight. LB medium with (open circles) or without (closed
circles) 0.3 mM IPTG was inoculated with diluted aliquots of the overnight
culture (starting O.D.600 = 0.05) and incubated at 37°C. Growth was
monitored by O.D. measurement.
B. Immunoblot analysis of PlsX in the PlsX-depleted strain NBS1014.
Each PlsX protein was detected using anti-PlsX antibodies and the
arrowhead indicates major non-specific bands. The cellular amount of SigA
as a control was also analysed with anti-SigA antibodies as a control for
loading.
C. Effect of PlsX depletion on cell morphology. NBS1014 cells were
collected for analysis after 1.5 hour cultivation with or without 0.3 mM
IPTG. Images show from left to right, FM4-64-stained membranes,
DAPI-stained DNA, and superposition of membranes (red) and DNA (blue).
Scale bar indicates 5 μm.
D. Effect of PlsX depletion on the cell length. Histograms show the
distribution of length under the indicated conditions. About 250 cells were
measured for each strain and time point.
E. The effect of PlsX depletion on the number of nucleoids per cell.
Histograms show the number of nucleoids per cell under the indicated
conditions.
Fig. S13. Aberrant formation of Z ring in the PlsX-depleted strain.
Subcellular localization of GFP-FtsZ (A) and FtsA-GFP (B). Images of
strains NBS1012 (A) and NBS1011 (B) show GFP-FtsZ or FtsA-GFP and
superposition of GFP (green) and DAPI-stained DNA (blue).
Cells were collected for analysis after 1.5 hours cultivation with or without
0.3 mM IPTG. Scale bar indicates 5 μm.
Fig. S14. Immunoblot analyses of GFP-FtsZ and FtsA-GFP in cells with or
without PlsX.
A. GFP-FtsZ and FtsA-GFP detected by immunoblot analysis in
PlsX-depleted strains (NBS1011 and NBS1012).
B. GFP-FtsZ and FtsA-GFP detected by immunoblot analysis in intact plsX
strains (NBS1372 and NBS1374) and plsX103 strains (NBS1373 and
NBS1375). Time (in hour) after the shift to 45oC is indicated.
In both A and B, each GFP-fused protein was detected using anti-GFP
antibodies and the arrowhead indicates the non-specific band. The asterisk
indicates the predicted size of free GFP. The cellular amount of SigA was
also analysed with anti-SigA antibodies as a control for loading.
Fig. S15. Effect of CCCP on the localization of GFP-PlsX.
Cellular localization of GFP-PlsX with (right) or without (left) the proton
ionophore 100 μM CCCP. Scale bar indicates 5 μm.
Fig. S16. Phenotype of the PlsY-depleted strain.
A. Effect of PlsY depletion on cell morphology. Cells of strain PMYNES
were collected for analysis after 2.5 hour cultivation with or without 1 mM
IPTG. From left to right, FM4-64-stained membranes, DAPI-stained DNA,
and superposition of FM4-64-stained membranes (red) and DAPI-stained
DNA (blue), respectively. White bar indicates 5 μm.
B. Growth profiles of the plsY-inducible strain PMYNES. The PMYNES
strain was streaked on LB pates containing 1 mM IPTG and incubated at
room temperature overnight. LB medium with (open circles) or without
(closed circles) 1 mM IPTG was inoculated with diluted aliquots of the
overnight culture (starting O.D.600 = 0.05) and incubated at 37°C. Growth
was monitored by O.D. measurement.
C. Effect of PlsY depletion on the cell length. Histograms show the
distribution of length under the indicated conditions. About 250 cells were
measured for each strain at each time point.
Fig. S17. Phenotype of the plsC temperature-sensitive mutant.
A. Effect of PlsC on cell morphology. Strains NBS1399 (intact plsC
(plsCspc)) and NBS1398 (temperature-sensitive plsC (plsC-C∆7)) cells
were collected for analysis at 0 and 1 h after the temperature was shifted to
49°C, which is the non-permissive temperature for plsC-C∆7. Images are
shown as in Fig. S16A.
B. Growth profiles of strains NBS1399 (closed circle) and NBS1398 (open
circle). These strains were grown at 30°C for 1 h in LB medium before the
temperature was shifted to 49°C. Growth was monitored by O.D.
measurement.
C. Effect of the plsC-C∆7 mutation on the cell length. Histograms show the
distribution of cell length in the indicated strains. Time after the shift to
49°C is indicated. About 250 cells were measured for each strain at each
time point.
Table S1 Bacterial strains and plasmids
Strain or plasmid
Genetic markers
Source/reference
Strains
Bacillus subtilis
168
trpC2
Laboratory stock
BFS1038
trpC2 ezrA::pMUTIN2.mcs (ermC)
Laboratory stock
NBS245
trpC2 ftsA::cat
This study
NBS367
trpC2 aprE::(PftsAZ-gfp-ftsZ cat)
YK065→168
NBS402
trpC2 PftsAZ-ftsA-gfp-cat
pFTSA8G→168
NBS800
trpC2 amyE::( Pxyl-gfp-plsX spc )
This study
NBS1009
plsX::pMHAcPLSX ( plsX-HA erm –Pspac-fabD-fabG)
This study
NBS1010
trpC2 plsX103 [D59G, L104S] spc
This study
NBS1011
trpC2 plsX::pMT3plsX (Pspac-plsX erm ) fabD::pfabD15(PrepU-neo-fabD-fabG )
NBS402→NBS1014
PftsAZ-ftsA-gfp cat
NBS1012
trpC2 plsX::pMT3plsX (Pspac-plsX erm ) fabD::pfabD15(PrepU-neo-fabD-fabG )
NBS367→NBS1014
aprE::(PftsAZ-gfp-ftsZ cat)
NBS1014
trpC2 plsX::pMT3plsX (Pspac-plsX erm ) fabD::pfabD15(PrepU-neo-fabD-fabG )
BYH12→168
NBS1327
trpC2 plsX spc
This study
NBS1328
trpC2 plsX [D59G] spc
This study
NBS1329
trpC2 plsX [L104S] spc
This study
NBS1330
trpC2
ASK510→168
NBS1341
trpC2 amyE::( Pxyl-gfp-plsX spc ) Pspac-ftsZ erm
NBS1330→NBS1008
NBS1342
minCD::tet
This study
NBS1359
trpC2 amyE::( Pxyl-gfp-plsX spc::cat )
pSpc::Cm→NBS800
NBS1362
trpC2 plsX::Pless-spc amyE::(Pxyl-gfp-plsX spc::cat)
This study
NBS1365
trpC2 minC::tet amyE::(Pxyl-gfp-plsX spc)
NBS1342→NBS800
NBS1371
trpC2 ftsA::cat amyE::(Pxyl-gfp-plsX spc)
NBS245→NBS800
NBS1372
trpC2 plsX spc amyE::(PftsAZ-gfp-ftsZ cat)
NBS1327→NBS367
NBS1373
trpC2 plsX103 [ D59G, L104S ] spc aprE::(PftsAZ-gfp-ftsZ cat)
NBS1010→NBS367
NBS1374
trpC2 plsX spc, PftsAZ-ftsA-gfp-cat
NBS1327→NBS402
NBS1375
trpC2 plsX103 [ D59G, L104S ] spc PftsAZ-ftsA-gfp cat
NBS1010→NBS402
NBS1398
plsC-C∆7 spc
unpublished strain
Pspac-ftsZ erm
NBS1399
plsC spc
unpublished strain
NBS1517
trpC2 plsX-his12 spc
This study
NBS1569
trpC2 thrC::( Phy-spank-ftsA erm )
This study
NBS1571
trpC2 thrC::( Phy-spank-mciZ erm )
This study
NBS1572
trpC2 thrC::( Phy-spank-sirA erm )
This study
NBS1576
spec-gfp-ftsA
SD100→168
NBS1578
trpC2 thrC::( Phy-spank-ftsA erm ) spec-gfp-ftsA
NBS1569→NBS1576
NBS1579
trpC2 thrC::( Phy-spank-ftsA erm ) amyE::( Pxyl-gfp-plsX spc )
NBS1569→NBS800
NBS1583
trpC2 thrC::( Phy-spank-mciZ erm ) amyE::( Pxyl-gfp-plsX spc )
NBS1571→NBS800
NBS1584
trpC2 thrC::( Phy-spank-sirA erm ) spec-gfp-ftsA
NBS1572→NBS1576
NBS1585
trpC2 thrC::( Phy-spank-sirA erm ) amyE::( Pxyl-gfp-plsX spc )
NBS1572→NBS800
NBS1875
trpC2 thrC::( Phy-spank-mciZ erm ) amyE::( Pxyl-gfp-plsX spc ) minCD::tet
NBS1342→NBS1583
NBS1876
trpC2 amyE::( Pxyl-gfpA206K-plsX spc )
This study
NBS1877
trpC2 thrC::( Phy-spank-gfp-plsX erm )
This study
NBS1878
trpC2 spec-cfp(Bs)-ftsA
This study
NBS1879
trpC2 spec-cfp(Bs)-ftsA amyE::( Pxyl-gfp-plsX spc::cat )
NBS1878→NBS800
NBS1880
trpC2 spec-cfp(Bs)-ftsA amyE::( Pxyl-gfp-plsX spc::cat ) thrC::( Phy-spanK-ftsA erm )
NBS1569→NBS1879
NBS1881
trpC2 spec-cfp(Bs)-ftsA amyE::( Pxyl-gfp-plsX spc::cat ) minCD::tet
NBS1342→NBS1879
ASK510
168 Pspac-ftsZ erm
K. Asai (unpublish)
PMYNES
plsY::pMutin [Pspac-plsY erm]
(Hara et al, 2008)
BYH12
plsX::pMT3plsX (Pspac-plsX erm ) fabD::pfabD15(PrepU-neo-fabD-fabG )
(Hara et al, 2008)
YK065
CRK6000 aprE::(PftsAZ-gfp-ftsZ cat)
Y.Kawai (unpublish)
SD100
spec-gfp-ftsA
S.Ishikawa (unpublish)
F‐ φ80lacZΔM15 Δ(lacZYAargF)U196 recA1 endA1 hsdR17 (rK‐ , mK+)
Invitrogen
Escherichia coli
DH5α
phoAsupE44 λ‐ thi1
Plasmids
pGFP-PlsX
bla amyE::Pxyl-gfp-plsX
spc
This study
pGFPA206K-PlsX
bla amyE::Pxyl-gfpA206K-plsX spc
This study
pHT001
amyE::Physpank cat
This study
pHT002
amyE::Physpank erm
This study
a
pHT003
thrC::Phy-spank erm amp
This study
pHT307
thrC::Phy-spank-mciZ erm amp
This study
pHT308
thrC::Phy-spank-gfp-plsX erm amp
This study
pHT402
thrC::Phy-spank-ftsA erm amp
This study
pHT408
thrC::Phy-spank-sirA erm amp
This study
pGFP7C
bla gfp cat
(Kuwana et al, 2006)
pFTSA8G
PftsAZ-ftsA-gfp cat
This study
pMHAc
bla erm Pspac-HA
K.Asai (unpublish)
pMHAcPLSX
bla erm Pspac-plsX (487-999bp)-HA
This study
pSpc::Cm
bla spc::cat
Laboratory stock
pDG1664
thrC::erm amp
(Guerout-Fleury et al, 1996)
pDR111
amyE::Physpank spc
(Ben-Yehuda et al, 2003)
pDR200
cfp amp
(Doan et al, 2005)
pSG1729
bla amyE'3 spc Pxyl-gfp amyE'5
(Lewis & Marston, 1999)
Abbreviations for antibiotic resistance are as follows: tet, tetracycline; spc, spectinomycin; cat, chloramphenicol; erm,
erythromycin; bla, ampicillin.
Table S2 PCR and mutational primers
Primer
Primer Sequence (5'-3')
Gene disruption
ftsA-11
GGTCAGGCCTTGAGGATC
ftsA-12
CGTTTGTTGAATTATCTATGGCACCTCCTCAC
ftsA-catFor
GTGCCATAGATAATTCAACAAACGAAAATTGGATAAAG
ftsA-catRev
TATCTATCTATTACTATAAAAGCCAGTCAT
ftsA-23
GGCTTTTATAGTAATAGATAGATAGTCATTCGGC
ftsA-24
CGAAGTACGTTATCCGCTTC
minCD-11
CCAGAAGGGCTGACAATCGG
minCD-12
TTCATAACCGTTAAATATTCACCTCAACAACATACTCATTTCG
minCD-tetFor
CGAAATGAGTATGTTGTTGAGGTGAATATTTAACGGTTATGAAGTGAAATTGA
minCD-tetRev
CTTTGTCAGATTCTTCTCTTTGATTCTATCACACATTATTACTAGAAATCCCTTTGAGAATG
minCD-23
AGGGATTTCTAGTAATAATGTGATAGAATCAAAGAGAAGAATCTGACAAAG
minCD-24
GGCTGTCTTTGCTGACTTCAAC
gfp fusion strain
plsXxhoI
CGCTCGAGCTGCCGGGACCGGAACTGCCGGGACCGGAAATGAGAATAGCTGTAGATGCAATGG
plsXecoRI
ACATGAATTCAAAACCTCCAGACTACTC
gfpmut1 A206K for
AAACTTTCGAAAGATCCCAACGAAAAGAGAG
gfpmut1 A206K rev
AGATTGTGTGGACAGGTAATGGTTG
PhygfpplsXF(NheI)
GCGCTAGCTCTAGAAAGGAGATTCCTAGGATGG
PhyplsXR(NheI)
GCGCTAGCCTACTCATCTGTTTTTTCTTCTTTCAC
ftsA up For
AGACGGATACGCCCTTGA
ftsA up rev
CACCTCGTTGTTCGATCATTTCTATTCTATTATTTGC
ftsA spc for
GAAATGATCGAACAACGAGGTGAAATCATGAG
ftsA spc rev
CTCCTTATGTCTAGGCCTAATTGAGAGAAG
cfp for
TTAGGCCTAGACATAAGGAGGAACTACTATGGTTTC
cfp rev
GCAGCGGAGCCAGCGGATCCTGCTGAGCCCTTATAAAGTTCGTCCATGCCAAGT
ftsA orf For
GGATCCGCTGGCTCCGCTGCTGGTTCTGGCATGAACAACAATGAACTTTACGTCAG
ftsA orf Rev
CCTGTCAGCACGAAGCC
Temperature-sensitive mutant
plsX-11
GTTCAGCCAGAAACAATCG
plsX-12
GCATGCTCCACCTTTATGAATG
plsX-mutFor
CATTCATAAAGGTGGAGCATGC
plsX-mutRev
CACCTCGTTGTTATCATCTGTTTTTCTTCTTTCAC
plsX-spcFor
AACAGATGAGTAACAACGAGGTGAAATCATGAG
plsX-spcRev
CTCCAGACTATTACTAGGCCTAATTGAGAGAAG
plsX-23
CAATTAGGCCTAGTAATAGTCTGGAGGTTTTTACATCATG
plsX-24
TCACAGGCGTCCAATACC
plsX-His12 strain
plsX-His12 ORF Rev
TTAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGCTCATCTGTTTTTTCTTCTTTCACTTC
plsX-His12 spc For
CACCACCACCACCACCACCACCACCACCACCACCACTAACAACGAGGTGAAATCATGAGC
Overexpression strain
PhyftsAF(SalI)
GCGTCGACACATAAGGAGGAACTACTATGAACAACAATGAACTTTACGTCAG
PhyftsAR(NheI)
GCGCTAGCTGACTATCTATCTATTCCCAAAACATGC
PhymicZF(HindIII)
GCAAGCTTCATACTAGAGCAAAAGGGGGC
PhymicZR(NheI)
GCGCTAGCTTATGGCTTTGAGATCCAATCTTTC
PhysirAF(SalI)
GCGTCGACACATAAGGAGGAACTACTATGGAACGTCACTACTATACG
PhysirAR (NheI)
GCGCTAGCTTAGACAAAATTTCTTTCTTTCACCGG
Resistant genes
cmRF
GCGAATTCTTCAACAAACGAAAATTGGATAAAG
cmRR
GCCACATGGTGTTACTATAAAAGCCAGTCAT
ermRF
GCGAATTCCGTGCTGACTTGCACCATATC
ermRR
GCCACATGGTGTTACTATTTCCTCCCGTTAAATAATAGATA
Specificity test of yeast two-hybrid analysis
cdsA-5Eco
GGGCGAATTCATGGTGGACATGAAACAAAGAATTTTGAC
cdsA-3Bam
GCGGATCCTGAAAAAAGGGCAAGCAGAAAGTAC
dgkA-5Eco
GCGAATTCGTGAATCGATTCTTCAAAAGCTTCG
dgkA-3Bam
GGGCGGATCCCAGCTTTGGCAAAAAAATAAGTAAACC
gpsA-5Eco
GCGAATTCATGAAAAAAGTCACAATGCTTGGAGCG
gpsA-3Bam
GGGCGGATCCCTTCACTTGATTTTCAAACGTATTTACC
pgsA-5Eco
GGGCGAATTCATGTCTAACTTACCAAATAAAATCACACTAGC
psd-5Eco
GGGCGAATTCATGTCTAACTTACCAAATAAAATCACACTAGC
psd-3Bam
GCGGATCCTTCTTCGTAACCTATCAGTTCTCCG
pssA-5Eco
GCGAATTCGTGAATTACATCCCCTGTATGATTACG
pssA-3Bam
GCGAATTCGTGAATTACATCCCCTGTATGATTACG
ugtP-5Bam
GGGCGGATCCTGAATACCAATAAAAGAGTATTAATTTTGACTGC
ugtP-3Sal
GCGTCGACCGATAGCACTTTGGCTTTTTGTTTGG
yfiX-N5Eco
GCGAATTCGTGCCGGGCGGCTTCGGCTCGTTTG
yfiX-C5Eco
GCGAATTCAAACCGATCGGAGAGAAAGCTG
yfiX-C3Bam
GCGGATCCGACGGAGTCTTTTTTGCTTTTGCC
yhdO-5Bam
GGGCGGATCCTGTATAAGTTTTGTGCAAATGCTTTAAAAG
yhdO-3Xho
GCCTCGAGTAGCTGATCAAGTTTATTCTCTAGTTC
ywnE-N5Eco
GCGAATTCGTGAGTATTTCTTCCATCCTTTTATCAC
ywnE-N3Bam
GCGGATCCTAGATACTCATCTCCGACGTTAAAG
ywnE-C5Eco
GCGAATTCGGGCTTAATCCGAAATTCGG
ywnE-C3Bam
GCGGATCCTAAGATCGGCGACAACAGCCG
plsX-5Eco
GCGAATTCATGAGAATAGCTGTAGATGCAATGG
plsX-3Bam
GCGGATCCCTACTCATCTGTTTTTTCTTCTTTCACTT
yneS-5Eco
GCGAATTCATGTTAATTGCTTTATTGATTATTTTGGC
yneS-3Bam
GCGGATCCTTATAACCATTTTACTTTAGGTTCTGTTTT
ezrA-N5Eco
GGGCGAATTCATGGAGTTTGTCATTGGATTATTAATTGTACTG
ezrA-N3Bam
GCGGATCCGGACGCTTCATCAATATCCAGTTC
ezrA-C5Eco
GCGAATTCGCAATCCTGCAGCTCATTGACG
ezrA-C3Bam
GCGGATCCAGCGGATATGTCAGCTTTGATTTTTTC
yshA/zapA5Eco
GGGCGAATTCTTGTCTGACGGCAAAAAAACAAAAACAACC
yshA/zapA3B
GCGGATCCATCCTTTTCTTTAAGCTGACGCTCC
dacA-5Eco
GGGCGAATTCTTGAACATCAAGAAATGTAAACAGCTACTG
dacA-3Bam
GCGGATCCAAACCAGCCGGTTACCGTATC
pbpD-N5Bam
GCGGATCCTGACCATGTTACGAAAAATAATCGGATGG
pbpD-N3Xho
GCCTCGAGCTGTACGTCCGCATACGGGAG
pbpD-C5Bam
GCGGATCCGCGGAGCGGCTGTGATTAATC
pbpD-C3Xho
GCCTCGAGATAAGCCGCTTGCAGCGTTC
pbpX-5Eco
GCGAATTCATGACAAGCCCAACCCGCAGAAG
pbpX-3Bam
GGGCGGATCCTTCTTGATTTAGAAGCTGGTATATTTTATTGTTCAC
ponA-N5Eco
GCGAATTCATGTCAGATCAATTTAACAGCCGTG
ponA-N3Bam
GCGGATCCAAATGCGCTGTATTTGTTTGTACTTGC
ponA-M5Eco
GCGAATTCGTTGAAGAGGTTATGAAGGAAATTGATG
ponA-M3Bam
GCGGATCCCATCGACATCGGCGATGAG
ponA-C5Eco
GCGAATTCACGGAAGCGGATCATTTGAG
ponA-C3Bam
GGGCGGATCCATTTGTTTTTTCAATGGATGATGAGTTTGTTTGTG
secA-N5Bam
GGGCGGATCCTGCTTGGAATTTTAAATAAAATGTTTGATCC
secA-N3Xho
GCCTCGAGACCGACTAGAACAGGCTGTC
secA-C5Bam
GCGGATCCCGGTTGCCGTTGAAACATC
secA-C3Xho
GCCTCGAGTTCAGTACGGCCGCAGCAATTTT
spoIIE-N5Bam
GCGGATCCTGGAAAAAGCAGAAAGAAGAGTGAAC
spoIIE-M5Bam
GCGGATCCAAGTGGCGAGATATATTCCGG
spoIIE-M3Xho
GCCTCGAGCATCATGCTGTAGCTGTCACCG
spoIIE-C5Bam
GCGGATCCAGCTTGGAGCCAGAAAATATGCTG
spoIIE-C3Xho
GGGCCTCGAGTGAAATTTCTTGTTTGTTTTGAAAGATTGCCG
divIB-5'
GCGAATTCATGAACCCGGGTCAAGACCG
divIB-3'
GCGTCGACGCCCCTCAATTTTCATCTTCC
divIB-C-5'Eco
GCGAATTCTCTGTTACAGGGAATGAAAATGTATC
divIB-3'
GCGTCGACGCCCCTCAATTTTCATCTTCC
divIC-5'
GCGAATTCTTGAATTTTTCCAGGGAACGAACG
divIC-3'
GCGGATCCGGCTACTTGCTCTTCTTCTCC
divIC-C-5'Eco
GCGAATTCACATCTTCCCTTAGTGCAAAAGAAG
divIC-3'
GCGGATCCGGCTACTTGCTCTTCTTCTCC
ftsA-5'
GCGAATTCATGAACAACAATGAACTTTACGTC
ftsA-3'
GCGGATCCTCCTCCTAATCTGCCGAATG
ftsE-5'
GCGAATTCATGATAGAGATGAAGGAAGTATATAAAGC
ftsE-3'
GCGGATCCTTAATCATATGAACCATACTCCCC
ftsL-5'
GCGAATTCATGAGCAATTTAGCTTACCAACCAG
ftsL-3'
GCGGATCCGGCATTTGAATCATTCCTGTATG
ftsL-C-5'Eco
GCGAATTCTGCGGCATATCAAACCAATATTGAGG
ftsL-3'
GCGGATCCGGCATTTGAATCATTCCTGTATG
ftsX-5'
GCGAATTCATGATTAAAATTCTCGGGCGCCAC
ftsX-3'
GCGGATCCCTTTATACTCGCAGAAACTTGCGG
ftsZ-5'
GCGAATTCATGTTGGAGTTCGAAACAAACATAGACG
ftsZ-3'
GCGGATCCTTGTCCTTTACATTAGCCGC
ylaO-5'
GCGAATTCATGTTAAAAAAAATGCTAAAATCTTATG
ylaO-3'
GCGGATCCTATCCTTCCCCTGTACACAC
B31-divIVA5Bam
GCGGATCCTGCCATTAACGCCAAATGATATTCAC
B31-divIVA3Pst
GCCTGCAGTTATTCCTTTTCCTCAAATACAGCG
B29-minC5Eco
GCGAATTCGTGAAGACCAAAAAGCAGCAATATG
B29-minC3Bam
GCGGATCCTCACATTCCTCCCTCAAGCC
B30-minD5Eco
GCGAATTCTTGGGTGAGGCTATCGTAATAAC
B30-minD3Bam
GCGGATCCATCACATTAAGATCTTACTCCG
B21-soj5Eco
GCGAATTCGTGGGAAAAATCATAGCAATTACG
B21-soj3Bam
GCGGATCCCTTTAGCCATTCGCAGCCAC
B24-pbpB5Bam
GCGGATCCATATTCAAATAACCGGAAAAGCGAACG
B24-pbpB3Sal
GCGTCGACCGACGGCTTTCTTTTTAATCAGG
B23-pbpF5Eco
GCGAATTCCATTATGTCATAGATGAAAAAAAG
B23-pbpF3Bam
GCGGATCCTTAAGAGGAAAACAATTTTGGCTGAACG
B26-yfhF5Bam
GCGGATCCTGAATATCGCAATGACGGG
B26-yfhF3SP
GCCTGCAGGTCGACTTATACAGTCTTTCGGTCCGC
B27-yfhK5Eco
GCGAATTCATGAAAAAGAAACAAGTAATGCTCGC
B27-yfhK3Bam
GCGGATCCTTATCGCATTTGTAAATCCTTTGTG
B28-yjoB5Bam
GCGGATCCTGACTAACATACCTTTCATTTATCAGTACG
B28-yjoB3Sal
GCGTCGACGCTTTCTTTTAGTGAAAACCGAC
pbpB-NF1Bam
GCGGATCCTGATTCAAATGCCAAAAAAGAATAAATTTA
B24-pbpB3Sal
GCGTCGACCGACGGCTTTCTTTTTAATCAGG
yppD-5Eco
GCGAATTCATGTCCGGTTATGTATGCAAG
yppD-3Bam
GCGGATCCTCAGCCGATGCGGTATACC
spsK-5'Eco
GCGAATTCTTGACAAAGGTATTGGTGACTGG
spsK-3'Bam
GCGGATCCTCAATCACACGCACTGCTC
References
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