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Supplemental Data

Bacterial Birth Scar Proteins Mark

Future Flagellum Assembly Site

Edgar Huitema, Sean Pritchard, David Matteson, Sunish Kumar Radhakrishnan, and Patrick H. Viollier

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Figure S1. Z-Ring Constriction Is Required for Localization of TipN-GFP and TipF-GFP to the Division Site

Early log phase cultures of tipF-gfp and tipN-gfp, treated with or without the FtsI

inhibitor cephalexin, were grown for 150 (left panels) and 480 min (right) before

being analyzed by DIC (left) and GFP (right) microscopy.

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Table S1. Analysis of Flagella in Wild Type, tipF and tipN Mutants by Transmission Electron Microscopy (TEM) Strain (Genotype)

SW Pole

ST Pole

ST Tip

ST Body

Cell Body

Total Cells w/ Flagella

Total Cells

NA1000 (wild type)

52 (100)

0

0

0

0

52 (31)

166

NR1751 (∆tipN)

205 (63)

29 (8)

29 (8)

61 (18)

6 (2)

326 (40)

824

NS142 (tipN::EZTn5)

58 (80)

2 (3)

3 (4)

9 (12)

1 (1)

73 (47)

155

NS250 (tipN::HyperMU)

54 (82)

4 (6)

3 (5)

5 (8)

0 66 (39)

170

NS267 (tipN::HyperMU)

42 (69)

2 (3)

3 (5)

6 (10)

8 (13)

61 (44)

137

NR1584 (∆tipF)

4 (100)

0 0 0 0 4 (1)

344

NS46 (tipF::Himar1)

2 (100)

0 0 0 0 2 (1)

167

NS187 (tipF::Himar1)

0 0 0 0 0 0 (0)

176

The number of cells is displayed for each mutant, and the percentages are shown in brackets.

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Table S2. Stalk Analysis in Wild Type, ∆tipF, ∆tipN and ∆tipN ∆tipF Mutants by TEM

Strain (Genotype)

Total Cells

Cells w/ Stalks

Bistalked (Pole/Pole)

Bistalked (Pole/Cell)

NA1000 (wild type)

100

50 (50)

0

0

NR1751* (∆tipN)

460 348 (76)

54 (15)

0

NR1584 (∆tipF)

180 146 (81)

1

0

NR1705 (∆tipN; ∆tipF)

745 575 (77)

88 (15)

9 (2)

*30% of the bistalked cells were unpinched and short (in the stalked cell stage or very early predivisional cell stage) Cell counts scored for the presence of two stalks. In parentheses are percentages. Note that there is a high incidence of stalks in the mutant cells possibly due to the filamentation defect associated with the loss of TipF and/or TipN. Stalked cells were subdivided into different classes with the percentages reflecting the fraction of the total cells with a stalk.

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Table S3. Localization of TipF-GFP in Wild Type and tipN Cells

Strain (Genotype)

Flagellar Pole

Multiple & Mislocalized Foci

Stalk Total Cells

NR1216 (NA1000; tipF-gfp) 49 1 0 50

NR1760 (∆tipN; tipF-gfp) 6 24 20 50

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Table S4. Localization of FliG-GFP in Wild Type (NA1000), ∆tipF and ∆tipN Cells

Strain (Genotype)

Flagellar Pole

Bipolar Foci at Cell Body

Stalk Total Cells

NR1744 (NA1000; xylX:: Pxyl-fliG-gfp)

47 51 2 0 100

NR1740 (∆tipF; xylX:: Pxyl-fliG-gfp)

3 3 10 3 100

NR1770 (∆tipN; xylX:: Pxyl-fliG-gfp)

13 55 23 9 100

DIC images as well as fluorescence images were used to score cells for localization of FliG-GFP to either the flagellated pole, both poles, the cell body, or the stalk.

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Table S5. CheA-GFP and PleC-GFP Localization in Wild Type (NA1000), ∆tipN and ∆tipF Mutant Strains Strain (Genotype)

Flagellar Pole

Bipolar Plane of Division

Stalk Total Cells

NR1212 (NA1000; cheA-gfp 97 3 0 0 100

NR1805 (∆tipF cheA-gfp 30 17 53 0 100

NR1806 (∆tipN; cheA-gfp) 26 12 62 0 100

PV760 (NA1000; pleC-gfp) 99 0 0 1 100

NR1885 (∆tipF; pleC-gfp) 97 5 0 15 127

NR1792 (∆tipN; pleC-gfp) 162 19 0 21 203

DIC images as well as fluorescence images were used to score cells for localization of CheA-GFP and PleC-GFP to either the flagellated pole, both poles, clustered near, or at the plane of cell division and accumulation in the stalk.

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Table S6. Localization of DivJ-GFP in Wild Type, ∆tipN and ∆tipF Mutant Strains Strain (Genotype)

Flagellar Pole

Bipolar Double Focus

Stalked Pole

Total Cells

LS3200 (NA1000; divJ-gfp) 0 2 3 95 100

NR1890 (∆tipF; divJ-gfp) 0 2 2 96 100

NR1891 (∆tipN; divJ-gfp) 0 2 5 93 100

Experimental Procedures

Strain Constructions

The ∆tipN, ∆tipF, ∆tipF194-420, ∆tipF194-202, and ∆tipF414-420 strains were

constructed by creating an in-frame deletion of the sequence encoding residues 24-

831 of TipN, 41-440, 194-420, 194-202 and 414-420 of TipF with plasmids pHP001,

pCWR207, pHP002, pHP003 and pHP004, respectively, using the standard two-step

recombination sucrose-counterselection procedure. To construct the ∆tipN ∆tipF

double mutant, the ∆tipN deletion was created in the ∆tipF mutant background. To

construct the tipN-gfp and tipF-gfp strains in which 3` end of the endogenous tipN or

tipF gene was replaced with one to which the gfp gene had been appended. Plasmids

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pSPX47 and pCWR169 that harbor the respective fragment and confer resistance to

kanamycin were integrated by a one-step homologous recombination into NA1000.

Plasmids pPxyl::ParB∆N40 (Figge et al., 2003) or pPxyl::ftsZ (also known as

pUJ142ftsZ) (Din et al., 1998) were mobilized into Caulobacter by intergeneric

conjugation from E. coli S17-1 (Ely, 1991). Exconjugants were isolated on PYE

plates containing nalidixic acid (20 µg/ml) and glucose (0.2%). tipN-gfp and tipF-gfp

cells containing pPxyl::ParB∆N40 or pPxyl::ftsZ were grown to log phase, washed and

re-suspended in PYEX (PYE supplemented with 0.3% xylose) or PYEG (PYE

supplemented with 0.2% glucose).

YB1585 (NA1000 ftsZ::Pxyl-ftsZ) (Wang et al., 2001) was transduced with lysates

from a strain bearing tipF-gfp or tipN-gfp allele marked with the apramycin resistance

gene aac(3)IV (Blondelet-Rouault et al., 1997). These strains were created by

ligating aac(3)IV on a SmaI fragment into the HpaI site of pSPX47 and pCWR169

and transforming the resulting plasmids into NA1000.

To make the CheA-GFP reporter strain, plasmid pCWR178 was introduced into

NA1000, selecting for kanamycin resistant transformants. The reporter was

subsequently transduced into NA1000, the ∆tipF and the ∆tipN mutant.

Chromosomal PleC-GFP and DivJ-GFP reporter constructs were transduced from

strain PV760 (Viollier et al., 2002) and LS3200 (Wheeler and Shapiro, 1999),

respectively, into NA1000, the ∆tipF and the ∆tipN mutant.

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NA1000 harboring a translational fljK-lacZ translational reporter, pfljK::lacZ

(Mangan et al., 1999), integrated at the fljK locus served as host for a phage lysate

made from the generalized transducing phage ΦCr30. This lysate was transduced into

NA1000 and the ∆tipF mutant. The transcriptional fljK-lacZ reporter plasmid, pfljK-

lacZ/290 (Wingrove et al., 1993), was transformed into NA1000 and the ∆tipF

mutant.

The ∆tipF mutation was introduced into the flbT650 (Johnson and Ely, 1979) mutant

using plasmid pCWR207.

Plasmids pCWR234 (tipF-H6) and pCWR235 (tipN-H6) were transformed into

NA1000, yielding strains NR1904 and NR1905, respectively. The resulting strains

were then transduced with a lysate from YB1585 and used in coimmunoprecipitation

studies described below.

The TipF-GFP and TipN-GFP reporter were introduced into strain CS606 for the

cephalexin experiments, yielding NR1382 and NR1371, respectively.

Plasmid Construction

KOD thermostable DNA polymerase (EMD Biosciences, San Diego, CA) or

PFU Turbo (Stratagene, La Jolla, CA) was used for amplifications using the

polymerase chain reaction. The tipF and tipN deletion construct was made in

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pNPTS138 (M.R.K. Alley, unpublished) through amplification of a 5′ 520 bp

fragment upstream from the tipF start codon and extending 120 bp into the predicted

open reading frame, and a 3′ region containing the last 36 bp codons of tipF and

extending 660 bp downstream of the gene. Amplification resulted in introduction of

BamHI and HindIII for the 5′ and BamHI and EcoRI sites for the 3′ fragment,

respectively. Similarly, a 599 bp 5′ fragment and an 810 bp 3′ fragment were

amplified from the tipN locus and used to delete residues 24–831 of TipN. The

appropriate pairs of fragments were restricted and ligated into EcoRI and HindIII

digested pNPTS138 vector to generate pCWRU207 (∆tipF) and pHP001 (∆tipN)

respectively. A pNPTS138 derivative harboring the tipF∆EAL allele to delete the

sequences encoding residues 194–420 that comprise the predicted EAL domain was

also constructed in an identical approach.

pSPX47 (tipN-gfp) and pCWR169 (tipF-gfp) were made by cloning the 3′ end of tipN

and tipF (nucleotides 2631–3199 of AE005823 and 2570–2001 of AE005746,

respectively) as XbaI/BamHI fragments into SpeI/BamHI-restricted pXGFP5 (M.R.K.

Alley, unpublished) and NheI/BamHI-restricted pXGFP4 (M.R.K. Alley,

unpublished) respectively. The resulting plasmids, pSPX47 and pCWR169, were

cleaved with HpaI and ligated to a SmaI fragment harboring aac(3)IV (Blondelet-

Rouault et al., 1997). To construct the Pxyl-fliG-gfp plasmid pCWR71, the fliG coding

sequence lacking the stop codon (nucleotides 4257–5276 of AE005767) was cloned

into pXGFP4 using NdeI/BamHI.

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To make pCWR178 (cheA-gfp) the 3′ end of the cheA gene (nucleotides 4486–5149

of AE005716) was amplified as XbaI/BamHI fragment and cloned into NheI/BamHI-

restricted pXGFP4. The resulting plasmid was cleaved with HpaI and ligated to a

SmaI fragment harboring aac(3)IV, yielding pCWR178.

Plasmids pCWR234 (tipF-H6) and pCWR235 (tipN-H6) were made by cloning PCR

fragments containing the 3′ end of tipF or tipN, respectively, into XbaI/EcoRI.-

restricted pHPV465 (Viollier et al., 2004). These fragments were amplified using the

same upstream primer as for pSPX47 and pCWR169 and a different downstream

primer fusing a sequence encoding HHHHHHGYKDDDDK-tag to the penultimate

codon of either tipN or tipF.

Construction of pCWR208 (Pxyl-tipF) and pCWR239 (Pxyl-tipFE211A) involved

several steps. The tipF coding sequence (nt 3356-1998 of AE5746) was PCR

amplified with primers that introduced an NdeI restriction site overlapping the start

codon (changed from TTG to ATG) and an EcoRI site after the stop codon. The

fragment was cloned into pOK12 (Vieira and Messing, 1991) using the restriction

sites in the primers, yielding pCWR206. The tipF fragment was excised using

NdeI/EcoRI and ligated along with the xylX promoter (Pxyl) fragment isolated from

pRW432 (R. Wright, unpublished) using SpeI/NdeI into the low-copy vector pLac290

(Wingrove et al., 1993) that had been restricted with XbaI and EcoRI. The resulting

plasmid, pCWR208, harboring Pxyl–tipF, was used in the complementation studies

with the ∆tipF mutant.

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The tipF(E211A) mutant allele was made by inverse PCR using pCWR206 as a

template along with primers that changed the codon for glutamate (GAG) at position

211 to one encoding alanine (GCG). The resulting plasmid was named pCWR226.

The mutant tipF allele was placed under Pxyl control in pLac290 using the same

strategy as the one used for construction of pCWR208. The resulting Pxyl -

tipF(E211A) plasmid was named pCWR239.

Analysis of the TipF and TipN Primary Amino Acid Sequence

The DAS (http://www.sbc.su.se/~miklos/DAS/maindas.html) and the Coils

(http://www.ch.embnet.org/software/COILS_form.html) server were used for

prediction of transmembrane segments and coiled-coils in TipF and TipN.

Expression of Wild Type TipF and TipF (E211A)

To test if the tipF(E211A) allele could correct the phenotypes of the ∆tipF

mutant, pCWR239 and pCWR208 were introduced into strain NR1584 and

cultivated in PYEG. Under these conditions, NR1584/pCWR208 swim, have polar

flagella, and secrete FljK and FlgE. Under the same conditions, NR1584/pCWR239

cells are nonmotile, lack external flagellar structures, and do not secret FljK and FlgE.

Transposon Mutagenesis and Isolation of tipN and tipF Mutants

To maximize genome-wide coverage of transposon mutagenesis, we used four

different mobile elements — (1) HyperMu <R6Kγori/KAN-1> (Epicentre, Madison,

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WI); (2) EZ-Tn5 <R6Kγori/KAN-2> (Epicentre); (3) the Himar1-derived mariner

transposon (Viollier et al., 2004); and (4) the Tn5 IS50L-derived ISlacZ/hah

transposon (Jacobs et al., 2003) containing an outwardly directed promoter to create

nonpolar insertions — collecting a library of ~17,000 mutants arrayed in 96-well

plates. This library was replica stamped on swarm agar plates to screen for mutants

with motility defects. A total of 194 mutants were identified, and the site of the

insertion of each transposon was mapped by first recovering HinP1I partially-

restricted neighboring chromosomal sequences as plasmids that transformed E. coli

EC100D-pir116 (Epicentre) to kanamycin resistance and then by sequencing across

the junction. Five mutants (NS46, NS142, NS187, NS250, NS267) were chosen for

further study.

References Blondelet-Rouault, M. H., Weiser, J., Lebrihi, A., Branny, P., and Pernodet, J. L. (1997). Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. Gene 190, 315-317. Din, N., Quardokus, E. M., Sackett, M. J., and Brun, Y. V. (1998). Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA. Mol Microbiol 27, 1051-1063. Ely, B. (1991). Genetics of Caulobacter crescentus. Methods Enzymol 204, 372-384. Figge, R. M., Easter, J., and Gober, J. W. (2003). Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol Microbiol 47, 1225-1237. Jacobs, M. A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., et al. (2003). Comprehensive transposon

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mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100, 14339-14344. Johnson, R. C., and Ely, B. (1979). Analysis of nonmotile mutants of the dimorphic bacterium Caulobacter crescentus. J Bacteriol 137, 627-634. Mangan, E. K., Malakooti, J., Caballero, A., Anderson, P., Ely, B., and Gober, J. W. (1999). FlbT couples flagellum assembly to gene expression in Caulobacter crescentus. J Bacteriol 181, 6160-6170. Vieira, J., and Messing, J. (1991). New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, 189-194. Viollier, P. H., Sternheim, N., and Shapiro, L. (2002). A dynamically localized histidine kinase controls the asymmetric distribution of polar pili proteins. Embo J 21, 4420-4428. Viollier, P. H., Thanbichler, M., McGrath, P. T., West, L., Meewan, M., McAdams, H. H., and Shapiro, L. (2004). From The Cover: Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. PNAS 101, 9257-9262. Wang, Y., Jones, B. D., and Brun, Y. V. (2001). A set of ftsZ mutants blocked at different stages of cell division in Caulobacter. Mol Microbiol 40, 347-360. Wheeler, R. T., and Shapiro, L. (1999). Differential localization of two histidine kinases controlling bacterial cell differentiation. Mol Cell 4, 683-694. Wingrove, J. A., Mangan, E. K., and Gober, J. W. (1993). Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter. Genes Dev 7, 1979-1992.