supplementary information: materials and methods: …
TRANSCRIPT
Radhakrishnan et al.
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SUPPLEMENTARY INFORMATION:
MATERIALS AND METHODS:
Plasmid constructions
The spmX deletion construct (pNPTS138-ΔspmXKO) was made by amplification
of two fragments. The first, a 558 bp fragment flanked by an EcoRI site at the 5`end and 5
a BamHI site at the 3` end, encompasses the upstream region of spmX and extends 21 nt
downstream of the predicted start codon. The second fragment, flanked by a BamHI site
at the 5`end and a HindIII site at the 3’end, harbored the last 21 bp of the spmX coding
sequence and extended 586 bp downstream of the gene. These two fragments were triple-
ligated into pNPTS138 that had been restricted with EcoRI and HindIII (M.R.K. Alley, 10
unpublished).
The coding sequences for SpmX (nt 6513-5218 from AE005889) and TacA
(nucleotides 9339-7873 of AE005993) were amplified and cloned as NdeI and EcoRI
restricted fragments into pET28a (EMD Biosciences, San Diego, CA), yielding
pCWR287 and pCWR322, respectively. The inserts were release by cleavage with NdeI 15
and EcoRI and cloned into XbaI/EcoRI-restricted plac290 along with the Pxyl promoter
fragment that was prepared as a SpeI/NdeI fragment to create pPxyl-spmX and pPxyl-tacA.
To make the PspmX-lacZ transcriptional reporter plac290-PspmX, nucleotides 6744-6354 of
AE005889 were amplified and ligated as EcoRI/BamHI fragment into plac290 to drive
transcription of the promoterless lacZ gene. 20
To construct pCWR321, an internal fragment of the tacA gene (encompassing
nucleotides 8468-8957 of AE005993) was amplified by PCR with primers adding an
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EcoRI and an XbaI restriction site at the 5`-end and 3`-end, respectively, and cloned into
SpeI/EcoRI-prepared pNPT228 (M.R.K. Alley, unpublished).
To create pMR20-pleC (also known as pHPV172) a 3.2 kbp fragment harboring
the entire pleC gene was released from pSCW408 (Crymes et al. 1999) using XhoI and
HindIII and ligated into pMR20 (Roberts et al. 1996). 5
Plasmid pPspmX-spmX was made by amplifying nucleotides 5218-7435 of
AE005889 with primers that introduced an XbaI and an EcoRI site at the 5`- and 3`-end
respective to the orientation of spmX and cloning the cleaved PCR fragment into plac290.
Plasmid pMT755 was made by PCR-amplifying the spmX gene with primers that
introduced an NdeI restriction site at the start codon and replaced the stop codon with a 10
deoxy-guanosine followed by a SacI restriction site. The resulting PCR product was cut
with NdeI and SacI and ligated into equally treated plasmid pXCHYC-2 (Thanbichler et
al 2007), thereby generating pMT638. A fragment comprising the 3’ portion of spmX
fused to the 5’ end of mCherry was isolated from pMT638 by restriction with XmnI and
BsrGI. It was then combined with a BsrGI/EcoRI-treated PCR fragment containing the 15
downstream flanking region of spmX and ligated into the allelic exchange vector
pNPTS138 (M. R. Alley, unpublished), which had been cut with EcoRV and EcoRI.
To construct pMT802 the spmX gene was PCR-amplified with primers that
introduced a BglII restriction site at the 5’ end and a SacI restriction site to the 3’ end.
After treatment with BglII and SacI, the PCR product was ligated into pXCHYN-2 20
(Thanbichler et al 2007), thereby fusing spmX in frame to the 3’ end of the mCherry
gene. The resulting plasmid (pMT776) was cut with NotI and NdeI and ligated with a
NotI/NdeI-cut PCR product containing the upstream flanking region of spmX. The
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resulting plasmid was cleaved with NotI and PflFI, and the fragment comprising the
partial 5` spmX`-mCherry-`spmX fragment was blunted with T4 DNA polymerase, and
ligated into EcoRV-treated pNPTS138.
The plasmids carrying spmX-mCherry alleles under control of the vanillate-
inducible promoter (Pvan) were made by generating PCR fragments with an NdeI site 5
overlapping the spmX start codon and having the stop codon replaced with an EcoRI site.
A codon-optimized mCherry fragment (synthesized by DNA. 2.0, Menlo Park, CA) was
cloned as an EcoRI/XbaI fragment behind Pvan on low-copy-number plasmid pRVMCS-
5 (Thanbichler et al. 2007) that had been restricted with EcoRI/NheI. The resulting
plasmid was named pCWR336. The mCherry fragment in pCWR336 carries a sequence 10
encoding an “EFGIHGGGGGV” linker peptide with an in-frame EcoRI site at the 5`end.
The XbaI site formerly located 3` of the stop codon of the mCherry fragment was
destroyed upon ligation into the NheI site of pRVMCS-5. Wild-type and mutant spmX
alleles encoding SpmX(1-150), SpmX(1-350) and SpmX(Δmur) (an in-frame deletion in
the sequence coding for E19-T34) in which an EcoRI site replaced the stop codon were 15
subsequently cloned into pCWR336 using NdeI and EcoRI. The resulting plasmids were
electroporated into NA1000 and ΔspmX mutant cells to observe the subcellular position
of the SpmX-mCherry derivatives. Variants harboring an EcoRI site after the stop codon
were made by PCR and subsequently cloned into pRVMCS-5 as NdeI/EcoRI fragments.
The resulting plasmids expressing untagged SpmX, SpmX(1-150), SpmX(1-350) and 20
SpmX(Δmur) were electroporated into the ΔspmX; divJ-yfp reporter strain to determine if
these variants could support localization of DivJ-YFP to the stalked pole.
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Strain Constructions
In-frame deletions of the sequence encoding residues 8-425 of SpmX were
created in strains NA1000, UJ506 (Aldridge et al. 2003), PV888 (Viollier et al. 2002),
UJ998 (Aldridge et al. 2003) using the standard two-step recombination sucrose-5
counterselection procedure induced by the pNPTS138-derived ΔspmX deletion plasmid
(pNPTS138ΔspmXKO) to yield the ΔspmX, ΔpleC ΔspmX, pleC(H610A) ΔspmX and
ΔdivJ ΔspmX strains, respectively. To localize DivJ, divJ-yfp and divJ-gfp alleles from
CJ826 (Lam et al. 2003), LS3200 (Wheeler and Shapiro 1999) were transduced into
NA1000, ΔspmX and ΔspmX ΔpleC strains. Strain LS3207 (pleC::Tn5 divJ-gfp) was also 10
from Wheeler and Shapiro (Wheeler and Shapiro 1999).
To visualize the subcellular position of DivK, plasmid pMR20divK-EGFP
(Jacobs et al. 2001) was electroporated into NA1000, ΔspmX and ΔspmX ΔpleC strains.
Alternatively, transductions were performed with lysates from strains CJ828 (divK-cfp)
or CJ833 (xylX::divK-cfp). Plasmid pXGFP4 (Pxyl-gfp) (M.R.K. Alley, unpublished) and 15
pCWR71 (Pxyl-fliG-gfp) (Huitema et al. 2006) were transformed into ΔspmX cells.
Fluorescence images of the resulting strains are shown in Supplementary Fig. S1.
Lysates from NS190 or NS349 were transduced into the ΔdivK strain harboring
pMR20divK-EGFP (Jacobs et al. 2001) by generalized transduction, yielding spmX
mutant strains in which the only version of DivK is DivK-GFP expressed from 20
pMR20divK-EGFP.
Strains MT237 (spmX-mCherry) and MT272 (mCherry-spmX) were made by
allelic exchange in NA1000 using pNPTS138-derived pMT755 and pMT802,
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respectively. MT237 was then transduced with lysates from CJ826 to create the spmX-
mCherry divJ-yfp strain NR3601.
Strain ΔpleC divKcs was made by creating an in-frame deletion in pleC in the
divKcs strain (Jacobs et al. 2001) using plasmid pPA43 (Aldridge et al. 2003). The ΔtacA
mutant used for immunoblotting was from Skerker et al (Skerker et al. 2005). In addition, 5
we engineered a tacA disruption mutant that was used as a host for β-galactosidase
measurements. This tacA mutant was made by transforming NA1000 cells to kanamycin
resistance with pCWR321.
The PspmX-lacZ transcriptional reporter strains were made by electroporation of
plac290-PspmX into NA1000, ΔpleC, NS217, NS229, tacA::pCWR321, divKcs, ΔpleC 10
divKcs, ΔdivJ, ΔpleC ΔdivJ and ctrA401 (Quon et al. 1996).
The plasmids expressing PleC(T614R) and PleC(F778L) have been described
previously (Matroule et al. 2004).
Co-immunoprecipitation 15
Mid log phase cells (50 ml) were harvested by centrifugation at 8500 g. Cells
were washed in buffer I (50 mM sodium phosphate, pH 7.4, 50 mM NaCl, 1 mM EDTA)
and lysed at room temperature for 15 min in buffer II (buffer I plus 10mM magnesium
chloride, 0.5% n-dodecyl-β-D-maltoside (Pierce, Rockford, IL), 1x protease inhibitors
[CompleteTM EDTA-free, Roche, Switzerland]) containing 1x Ready-Lyse lysozyme 20
solution (Epicentre Technologies, Madison, WI) and 30 units of DNase I (Roche).
Cellular debris was removed by centrifugation at 10,000 g for 3 min at 4oC. The
supernatant was pre-cleared with 30 μl of Protein-A or Protein-G agarose beads (Roche).
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To the pre-cleared solution, rabbit polyclonal anti-SpmX antibodies (1:500 dilution) or
mouse monoclonal anti-GFP antibodies [Living colors® A.v. Monoclonal Antibody (JL-
8), Clontech, Mountain View, CA] (1:300 dilution) was added and incubated for 90 min
at 4oC. The antibody-protein complexes were trapped using Protein-A and Protein-G
agarose beads, respectively, washed once with buffer I plus 0.5% n-dodecyl-β-D-5
maltoside, twice with 1% IP buffer (Protein G Immunoprecipitation kit Sigma-Aldrich,
St. Louis, MO), once with 0.1% IP buffer in order to remove salts and finally
resuspended in 70 μl of 1x Laemli sample buffer. After boiling and centrifugation,
precipitated proteins were identified by immunoblotting using monoclonal antibodies to
GFP (to detect DivJ-YFP), or polyclonal antibodies to SpmX or McpA. 10
Quantitative Chromatin Immunoprecipitation (qChIP) Assays
Mid-log phase cells were cross-linked in 10mM sodium phosphate (pH 7.6) and
1% formaldehyde at room temperature for 10 min and on ice for 30 min thereafter,
washed thrice in phosphate buffered saline (PBS) and lysed in a Ready-Lyse lysozyme 15
solution (Epicentre) according to the manufacturer`s instructions. Lysates were sonicated
(Sonicator 3000, Misonix Inc., Farmingdale, NY) on ice using 10 bursts of 20 sec at
output level 4.5 to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared
by centrifugation at 14,000 rpm for 2 min at 4oC. Lysates were normalized by protein
content, diluted to 1 ml using ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM 20
EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl plus protease inhibitors (Roche) and
pre-cleared with 80 μl of protein-A agarose (Roche), 100μg BSA and 300μg herring
sperm DNA. Ten % of the supernatant was removed and used as total chromatin input
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DNA. Anti-CtrA or anti-TacA antibody was added to the rest (1:1,000 dilution),
incubated overnight at 4oC with 80 μl of protein-A agarose beads pre-saturated with
BSA-Herring sperm DNA, washed once with low salt buffer (0.1% SDS, 1% Triton X-
100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1%
SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl) and 5
LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM
Tris-HCl (pH 8.1) and twice with TE buffer (10 mM Tris-HCl (pH 8.1) and 1 mM
EDTA). The protein•DNA complexes were eluted in 500 μl freshly prepared elution
buffer (1% SDS, 0.1 M NaHCO3), supplemented with NaCl to a final concentration of
300 mM and incubated overnight at 65oC to reverse the crosslinks. The samples were 10
treated with 2 μg of Proteinase K for 2 h at 45oC in 40 mM EDTA and 40 mM Tris-HCl
(pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1),
ethanol-precipitated using 20 µg of glycogen as carrier and resuspended in 50 μl of
water.
15
Real-time PCR
Real-time PCR was performed using a MyIQ single color real-time PCR detection
system (Bio-Rad, Hercules, CA) using 5% of each ChIP sample, 12.5 μL of SYBR green
PCR master mix (Bio-Rad), 10 pmol of primers and 5.5 μl of water per reaction.
Standard curve generated from the cycle threshold (Ct) value of the serially diluted 20
chromatin input was used to calculate the percentage input value of each sample. Average
values are from triplicate measurements done per culture. The final data was generated
from three independent cultures. The DNA regions analyzed by real-time PCR were from
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nucleotide -237 to +6 relative to start codon of spmX, from -287 to -91 relative to the start
codon of pilA, from +313 to +32 relative to the start codon of fliL, from -226 to +30
relative to the start codon of tacA, from -143 to +24 relative to the start codon of CC0167
and from -145 to +34 relative to the start codon of CC1695.
5
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SUPPLEMENTARY FIGURES:
Figure S1. Complementation of the ΔspmX phenotype. Expression of full length
spmX under the control of its native promoter from a low copy plasmid (pPspmX-spmX) 5
complements the DivJ-YFP localization defect (A) and the motility defect of the ΔspmX
mutant (B). The vector alone does not restore these functions.
Figure S1
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Figure S2. Aberrant cell division of ΔspmX cells.
(A) FliG-GFP expressed from xylose inducible promoter (Pxyl) is localized to the
constriction sites in the ΔspmX mutants grown in PYE containing 20 mM xylose. This is
consistent with the visualization of flagella at constriction sites by TEM. FliG is part of 5
the flagellar assembly apparatus, forming the C-ring/switch complex at the cytoplasmic
membrane (Macnab 2003). (B) Expression of GFP from Pxyl shows ΔspmX mini cells
exhibiting cytoplasmic fluorescence. Yellow arrow heads indicate the aberrant cell
division sites to which FliG-GFP is localized (A) and that constrict mini cell
compartments (B). 10
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Figure S3. DivK and DivJ are delocalized in spmX mutants.
(A) DivK-GFP is delocalized in the spmX mutant when the only source of DivK is
from a plasmid expressing DivK-GFP under control of its native promoter. (B)
Immunoblots show that disruption of spmX has no effect on the steady-state levels of
DivK-GFP or DivK. (C) DivJ-YFP and (E) DivJ-GFP are not localized in the ΔspmX 5
mutant cells. Immunoblots confirm that steady-state levels of (D) DivJ-YFP and (F)
DivJ-GFP are not affected in spmX mutants. X and Y denote cross-reacting bands.
10
Figure S4. Cell-cycle accumulation of SpmX-mCherry.
A blot harboring cell extracts from spmX-mCherry cells at different stages in the
cell was probed with antibodies to SpmX and CtrA. 15
Figure S4
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5
Figure S5. Co-immunoprecipitation of DivJ-YFP and SpmX.
The upper panel shows the same blot as in Fig. 4H. The lower panel shows
immunonanalysis of the same blot re-probed with antibodies to the McpA chemoreceptor.
10
Figure S5
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5
Figure S6. SpmX and PilA accumulation require PleC phosphatase activity.
Immunoblot analysis of SpmX, PilA and CtrA (control) steady-state levels in
NA1000, ΔpleC and ΔpleC strains harboring plasmids encoding PleC(T614R) or 10
PleC(F778L). While the former (K-P-) lacks both kinase (K) and phosphatase activity (P),
the latter (K-P+) can still function as a phosphatase (Matroule et al. 2004).
Figure S6
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Figure S7. qChIP analysis of CtrA and TacA occupancy at promoters in vivo.
(A) Precipitation of spmX promoter DNA in wild type cells using anti-CtrA
antibody is very inefficient compared to other known CtrA-dependent promoters such as
pilA, which contains multiple CtrA boxes, fliL or tacA (promoters with one CtrA box). 5
Figure S7
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(B) The spmX promoter, but neither the CC0167 nor the CC1695 promoter, two
other uncharacterized PleC-dependent genes (Chen et al. 2006), were precipitated
efficiently with the anti-TacA antibody from wild type lysates.
5
Figure S8. TacA expression induces stalk formation in the pleC::Tn5 mutant.
Expression of tacA from a xylose-inducible promoter on a low copy vector (pPxyl-10
tacA) restores stalk synthesis in pleC::Tn5 mutants. Cells containing the empty vector are
stalkless. White arrowheads indicate stalks.
Figure S8
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Supplementary Table S1. 5
Stalk analysis in wild type, spmX and divKcs mutants by TEM. Cell counts scored for the presence of two stalks. Values in at room temperature for 15 min parenthesis are
percentages.
Genotype
# late PD cells w/ bipolar
stalks
# Fil. cells w/ bipolar
stalks
wild type
0/100
0/100
divKcs
11/121 (9%)
17/98 (17%)
ΔspmX
50/180 (28%)
31/132 (23%)
10
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