Table 1.Plasmids used in this study
Plasmids Relavantcharacteristics or construction Source or reference
pBXMCS-2 High-copy replicating plasmid for xylose-inducible gene expression,kanR Thanbichler et al (2007)
pBW005 pRVMCS-5 with clpX* inserted between NdeIand NheIsites This Study
pBW015 pVGFPC-1 with clpX inserted between NdeIand PacIsites This Study
pBW016 pXMCHYC-4 with ftsZ inserted between NdeIand PacIsites This Study
pBW020 pXSTZN-2 with ftsZ inserted between KpnIand SacIsites This Study
pBW030 pRVGFPC-5 with clpA inserted between NdeIand PacIsites This Study
pBW041 pBXMCS-2 with ftsA inserted between NdeIand PacIsites This Study
pBW045 pXSTZN-2 with ftsZDDinserted between KpnIand SacIsites This Study
pBW047 pXSTZC-2 with ftsZ inserted between NdeIand AgeIsites This Study
pBW048 pXSTZC-2 with ftsA inserted between NdeIand AgeIsites This Study
pBW062 pET21B with tap-ftsA1236-1326(MTS) insertfrom pBW061 inserted in between NdeIand SacIsites This Study
pEG032 pXSTZN-2 with ftsQ inserted between KpnIand AgeIsites Goley ED,unpublished
pEG033 pXSTZN-2 with ftsA inserted between KpnIand SacIsites Goley ED,unpublished
pET21a Bacterial vector for expressing N- and/or C-terminal His6-tagged proteins in E.coli Novagen
pET21b Bacterial vector for expressing N- and/or C-terminal His6-tagged proteins in E.coli Novagen
pMT219 pET21a with ftsZ inserted between NdeIand SacIsites Thanbichler et al (2006)
pRVGFPC-5 Low-copy replicating plasmid for vanillate-inducible C-terminal eGFPfusions,TetR Thanbichler et al (2007)
pRVMCS-5 Low-copy replicating plasmid for vanillate-inducible gene expression,TetR Thanbichler et al (2007)
pVGFPC-1 Low-copy replicating plasmid for vanillate-inducible C-terminal eGFPfusions,StrepR/SpecR Thanbichler et al (2007)
pXMCHYC-4 Integrating plasmid for generation ofC-terminal mCherry fusions atxylX locus,GentR Thanbichler et al (2007)
pXSTZC-2 Integrating plasmid for generation ofC-terminal TAPtag fusions atxylX locus,KanR Goley ED,unpublished
pXSTZN-2 Integrating plasmid for generation ofN-terminal TAPtag fusions atxylX locus,KanR Goley ED,unpublishedTab l e  2 .  Caulobacter crescentus strains used in this study
Strains Relevantgenotpye Construction or Source 
LS101 CB15N - synchronizable derivative ofwild-type strain CB15 Evinger and Agabian (1997)
LS5344 CB15N vanA::PvanA-clpX-egfp pBW015 into LS101
LS5345 CB15N vanA::PvanA-clpX-egfp, xylX::PxylX-ftsZ-mCherry pBW016 intoLS5344
LS5346 CB15N ftsZ::ftsZ∆C xylX::PxylX-ftsZ, vanA::PvanA-clpX-egfp pBW015 into YB1585
LS5347 CB15N xylX::PxylX-tap-ftsZ pBW020 into LS101
LS5348 CB15N xylX::PxylX-tap-ftsA pBW033 into LS101
LS5349 CB15N pPvanA-clpX* pBW005 into LS101
LS5350 CB15N pPvanA-clpX*, xylX::PxylX-tap-ftsZ pBW020 into LS5349
LS5351 CB15N pPvanA-clpX*, xylX::PxylX-tap-ftsA pEG033 into LS5349
LS5352 CB15N pPvanA-clpX*, xylX::PxylX-tap-ftsQ pEG032 into LS5349
LS5353 CB15N xylX::PxylX-ftsZ-tap pBW047 into LS101
LS5354 CB15N xylX::PxylX-tap-ftsZ-DD (TAP-FtsZA507D N508D) pBW045 into LS101
LS5355 CB15N xylX::PxylX-ftsA-tap pBW048 into LS101
LS5356 CB15N ∆clpA::Ω, xylX::PxylX-tap-ftsZ pBW020 into UJ838
LS5357 CB15N ∆clpA::Ω, xylX::PxylX-tap-ftsA pEG033 into UJ838
LS5358 CB15N ∆clpA::Ω, xylX::PxylX-tap-ftsQ pEG032 into UJ838
LS5359 CB15N pPvanA-clpX*, pPxylX-ftsA pBW041 into LS5349
LS5360 CB15N pPvanA-clpA-egfp pBW030 into LS101
LS5361 CB15N ∆clpA::Ω, pPvanA-clpA-egfp pBW030 into UJ838
LS5362 CB15N ∆clpA::Ω ftsZ::ftsZ∆C xylX::PxylX-ftsZ Transduction of∆clpA::Ω SpecR into YB1585
LS5363 CB15N ftsZ::ftsZ∆C xylX::PxylX-ftsZ pPvanA-clpX* pBW005 into YB1585
LS5364 CB15N pPxylX-ftsA pBW041 into LS101
LS5365 CB15N ∆clpA::Ω pPxylX-ftsA pBW041 into YB1585
LS5366 CB15N pPvanA-clpX*, xylX::PxylX-ftsA-tap pBW048 into LS5349
LS5367 CB15N ∆clpA::Ω, xylX::PxylX-ftsA-tap pBW048 into UJ838
UJ838 CB15N ∆clpA::Ω Grunenfelder et al (2004)
YB1585 CB15N ftsZ::ftsZ∆C xylX::PxylX-ftsZ Wang et al (2001)      0 hr      6 hr      0        30        60        90        120      150 min. 
α-FtsZ α-FtsZ
A B
Supplementalry Materials 
 
Fig. S2. Intracellular levels of FtsZ. (A) Immunoblots of samples taken concurrently with the fluorescence microscopy. A 
sample of cells from a mixed population of the strain ftsZ::ftsZ C xylX::PxylX-ftsZ PvanA-clpX-egfp grown in minimal media 
supplemented with 0.3% xylose was taken before the population of cells was washed and resuspended in minimal media 
lacking xylose. A sample of cells was subsequently taken again after growth in the absence xylose for six hours. Each 
sample was then subjected to immunoblot analysis with anti-sera specific for FtsZ. (B) FtsZ levels upon reinduction with 
xylose after a six-hour depletion. A population of cells from the same population used above was resuspended in media 
supplemented with 0.3% xylose. A sample of cells was taken every 30 minutes (starting with t = 0) and subjected to 
immunoblot analysis with antisera specific to FtsZ.  
 
 
Supplementary Figures
- +
Vanillate
ɑ-CtrA
ɑ-ClpX
 
Fig. S3. Levels of CtrA and ClpX in the presence or absence of vanillate. (A) Cells from the strain pPvanA-clpX* were grown 
in rich PYE media in the presence of the vanillate inducer for two hours. Aliquots were taken before and after the addition 
of vanillate and subjected to immunoblot analysis using CtrA or ClpX-specific antiserum. 
ɑ-GFP
- +
B
Vanillate
            0            20            40            60           80           100         120         140 min.
CB15N
∆clpA
    ∆clpA
ClpA-GFP
A
 
 
Swarmer Stalked
Predivisonal
Fig. S1. Intracellular levels of Flif and ClpA-GFP. (A) ClpA-GFPrescues cell cycle dependent degradation of the FliF ClpA
substrate. Swarmer cells from strains CB15N wild type, ΔclpA or ΔclpA: pPvanA-clpA-egfp induced with 50 mM vanillate for 
two hours, were isolated and allowed to progress synchronously through the cell cycle. Aliquots were taken at 20-minute 
intervals and subjected to immunoblot analysis using FliF-specific antiserum. (B) Intracellular levels of ClpA-GFP. Cells of 
the strain ∆clpA pPvanA-clpA-gfp were grown to exponential phase in M2G medium, induced with 0.5 mM vanillate for 2 h, 
and analyzed by immunoblotting using anti-GFPantiserum (+ van). As a control, uninduced cells were subjected to the 
same analysis (- van).  
Fig. S5 . Cl pX N - t erminal domain is  import ant  for prot eolys is  of F t s Z . R epres ent at ive images  of react ions  s howing purified 
Cau lobac ter F t s Z  in t he pres ence of Cl pXP or Cl pX( N 61)P and AT P i n vit ro. R eact ion condit ions  cons is t  of 1 µ M Cl pX 6 or 
1 µ M Cl pX( N 61)6, 1 µ M Cl pP14 and 1 µ M F t s Z . 
0 30 60 90 120 min.
FtsZ
ClpX
ClpX∆N61
0 20 40 60 80 100 120 min.
ck
FtsA ∆1-18, ∆MTS
A B
0 15 30 45 60 75 90 min.
ClpP
T AP-FtsA MTS
ClpX/ck
FtsA ∆1-18, ∆MTS
0- 30 60 90 120 150 min.
0 15 30 45 60 75 90 min.
ClpX/ck
ClpP
T AP-FtsA MTS
ClpAP ClpXP
 -  ClpX*  (t 1/  2  = 44.55min± 2.81min)
+ ClpX* (t 1/2  = 37.22 min ± 0.14 min)
-  van
+ van
 0   15   30  45   60  75    90  105 120 135 min.
A
0 50 100 150
1.0
10.0
100.0
T ime (min.)
%
R
el
at
iv
e
B
an
d
In
te
ns
ity
 (
A
.U
.)
+ clpA (t 1/2  = 41.99min± 2.39min)
 -  clpA (t 1/2  = 50.29 min ± 3.49 min)
 0   15   30  45  60  75  90 105 120 135 min.
 -  clpA
+ clpA
B
0 50 100 150
1.0
10.0
100.0
T ime (min.)
%
R
el
at
iv
e
B
an
d
In
te
ns
ity
 (
A
.U
.)
Fig. S4. FtsQ is not a substrate of ClpXPand ClpAP in vivo. (A) Levels of TAP-FtsQ in the presence or absence of ClpX*. 
ClpX* is a dominant, catalytically inactive mutant of ClpX. Cells from the strain pP vanA-clpX* xylX::PxylX-tap-ftsQ were grown in 
rich PYE media in the presence of vanillate and xylose for two hours. Cells were then washed and resuspended in media 
lacking xylose. Aliquots were taken at 15-minute intervals and subjected to immunoblot analysis using TAP-specific antise-
rum in the presence or absence of the vanillate inducer. (B) Levels of TAP-FtsQ in the presence or absence of ClpA. Cells 
from the strain ΔclpA::Ω xylX::PxylX-tap-ftsQ were grown in rich PYE media in the presence of xylose for two hours, washed 
and resuspended in media lacking xylose. Aliquots were then taken at 15-minute intervals and subjected to immunoblot 
analysis using TAP-specific antiserum in the presence or absence of clpA allele. A graph of the relative band intensity as a 
function of time after the cessation of TAP-FtsQ synthesis is shown below each immunoblot.
Fig. S6. The N- and C-terminus of FtsA is important for proteolysis by ClpAPand ClpXP. (A and B) Representative images of 
reactions showing purified FtsA ∆1-18, ∆MTS or TAP-FtsA MTS in the presence of ClpAP or ClpXP and ATP in vitro. Reaction 
conditions consist of 1µM ClpA 6 or 1µM ClpX 6, 1µM ClpP 14 and 1µM FtsA ∆1-18, ∆MTS (or 1.5 FtsA ∆1-18, ∆MTS in Fig. S6B) and 1 µM 
TAP-FtsA MTS.