E. coli plasmids.
Relevant plasmids are listed in Table . The plasmids pET21b (Novagen), pGAD-C1 and pGBDU-C1 (42
), pDR10 (36
), pDB361 and pDR120 (37
), pCH151 (10
), pKNT25 (45
), pCH235 (6
), pCH363, pEZ1, pTB97, pTB98, pTB146, and pTB183 (7
), and pMG20 (29
) have been described before.
Unless indicated otherwise, TB28 chromosomal DNA was used as a template in amplification reactions. Sites of interest (e.g., relevant restriction sites or those engineered for targeted recombination) are underlined in primer sequences.
To construct pBL3 (attHK022 Plac::zapC-le), the 1,339-bp ApaI-HindIII fragment of pCH315 (see below) was used to replace the 1,849-bp ApaI-HindIII fragment of pTB183 (attHK022 Plac::gfp-zapA).
For pBL4 (attHK022 Plac::zapC-gfp), the 2,103-bp ApaI-HindIII fragment of pMG6 (see below) was used to replace the 1,849-bp ApaI-HindIII fragment of pTB183 (attHK022 Plac::gfp-zapA).
For pBL31 (PBAD::zapC-le), the 1,017-bp XbaI-XhoI fragment of pMG20 [PBAD::sstorA-bfp-ftsN(71-105)-le] was replaced with the 582-bp XbaI-XhoI fragment of pCH315.
To obtain pCH299 (PT7::zapC-h), the zapC gene of pMG6 was amplified with the primers 5′-GAGGCATATGCGAATTAAACCAGACGATAACTG-3′ and 5′-GTCAGCTCGAGGACTGCCTGTTCGAGGCTGAAGC-3′. The product was digested with NdeI and XhoI, and the 542-bp fragment was used to replace the 77-bp NdeI-XhoI fragment of pET21b.
For pCH315 (Plac::zapC-le), the 582-bp XbaI-XhoI fragment of pCH299 was used to replace the 512-bp XbaI-XhoI fragment of pCH235 (Plac::mreD-le).
To obtain pCH320 (PADH1::gal4BD-zapC) and pCH321 (PADH1::gal4AD-zapC), the zapC gene of pMG6 was amplified with the primers 5′-GGTAGGATCCATGCGAATTAAACCAGACGATAACTG-3′ and 5′-GGCGGTCGACTTAGACTGCCTGTTCGAGGCTGAAGC -3′. The product was digested with BamHI and SalI, and the 549-bp fragment was used to replace the 12-bp BamHI-SalI fragments of pGBDU-C1 (pCH320) and pGAD-C1 (pCH321), respectively.
For pCH322 (PT7::h-sumo-zapC), the zapC gene of MG1655 was amplified in two reactions using the primers 5′-GGTATGCGAATTAAACCAGACGATAACTGGCG-3′ or 5′-ATGCGAATTAAACCAGACGATAACTGGCG-3′ and 5′-AGTTCTCGAGTTAGACTGCCTGTTCGAGGCTGAAGC-3′. The products were mixed, heated to render single-stranded DNA (ssDNA), cooled to allow strands to reanneal, and treated with XhoI. The 547-bp product was then used to replace the 41-bp SapI-XhoI fragment of pTB146 (PT7::h-sumo-).
The plasmid pCH372 [PT7::h-sumo-zapC(L22P)] was made similarly to pCH322 except that pWM3632 [Ptrc::zapC(L22P)-gfp] was used as a template.
The plasmids pCH373 [PADH1::gal4AD-zapC(L22P)] and pCH374 [PADH1::gal4BD-zapC(L22P)] were obtained as described above for pCH321 and pCH320, except that pWM3632 was used as a template.
To create pCH438 [attHK022 Plac::zapC(L22P)-gfp], the 205-bp SapI-BsmI fragment of pWM3632 was used to replace the equivalent fragment of pBL4.
The plasmid pCH458 [Plac::zapC(L22P)-le] was obtained in two steps. First, the 542-bp NdeI-XhoI fragment of pCH438 was used to replace the 77-bp NdeI-XhoI fragment of pET21b, yielding pCH457 [PT7::zapC(L22P)-h]. The 582-bp XbaI-XhoI fragment of pCH457 was subsequently used to replace the 512-bp XbaI-XhoI fragment of pCH235 (Plac::mreD-le).
For pJE20 (PADH1::gal4AD-ftsZ), the 1,163-bp BamHI-SalI fragment of pDR10 (PT7::hfkt-ftsZ) was used to replace the 12-bp BamHI-SalI fragment of pGBDU-C1, yielding pJE15 (PADH1::gal4BD-ftsZ). The 1,163-bp BamHI-SalI fragment of pJE15 was next used to replace the 12-bp BamHI-SalI fragment of pGAD-C1.
For pMG6 (Plac::zapC-gfp), MG1655 chromosomal DNA was used as a template to amplify the zapC gene with the primers 5′-TGTTTCTAGATTGTTGAGGTTATTAAGCGAAGCGAC-3′ and 5′-GTCAGCTCGAGGACTGCCTGTTCGAGGCTGAAGC-3′. The product was digested with XbaI and XhoI, and the 616-bp fragment was used to replace the 1,026-bp XbaI-XhoI fragment of pCH151 (Plac::zipA-gfp).
To obtain pTB198 (attλ Psyn1::gfp-ftsZ), the 1,936-bp XbaI-SalI fragment of pDR120 (Plac::gfp-ftsZ) was used to replace the 1,109-bp XbaI-SalI fragment of pEZ1 (attλ Psyn1::gfp-zapA).
For pWM2978 (Ptrc::zapC-gfp), W3110 chromosomal DNA was used as a template to amplify zapC with the primers 5′-AAAGAGCTCCGAATTAAACCAGACGATAACTGGC-3′ and 5′-TTTTCTAGAGACTGCCTGTTCGAGGCTGAAGC −3′. The product was digested with SacI and XbaI, and the 539-bp fragment was used to replace the 17-bp SacI-XbaI fragment of pDSW208, placing zapC downstream of a ribosome binding site on the vector.
The plasmid pWM3632 [Ptrc::zapC(L22P)-gfp] was obtained in a manner similar to that of pWM2978, as described below.
Isolation of zapC mutant alleles.
Using the same template, primers, and cloning strategy as described for pWM2978 (Ptrc::zapC-gfp) above, the zapC gene was amplified with Taq polymerase to increase the chances of mutation. The ligation mixture was transformed into TX3772, and cells were plated on LB plus ampicillin (LB-Amp) agar supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Because the expression of zapC-gfp from pWM2978 was sufficiently toxic to prevent colony formation under these conditions, any colonies that did grow were candidates to harbor a plasmid encoding impaired ZapC. Several colonies grew, and plasmids from these colonies were transformed back into TX3772 under the same selection conditions. Those that gave rise to transformants at high efficiency were then subjected to sequencing of the putative zapC mutant. Two loss-of-function mutants of zapC were identified, encoding a L22P mutant and a E72G/R164A double mutant. Both encoded stable proteins, and they had similar phenotypes. The L22P mutant (encoded on pWM3632) was chosen for further study.
Relevant E. coli strains are listed in Table .
E. coli strains used in this study
Strains BL5 and BL6 were obtained by P1-mediated transduction (hereinafter referred to simply as “transduction”) of zapCSlm260 (zapC::EZTnKan-2) from Slm260 to TB28 and TB43, respectively.
CH41 was obtained by transduction of zapA<>cat from CH21 to TB28. (The symbol “<>” denotes DNA replacement by recombineering.)
CH56 was created by λRed-mediated recombineering (16
). The cat
cassette of pKD3 was amplified using the primers 5′-GTACTTTTATTGTTGAGGTTATTAAGCGAAGCGACAATGGATT
CATATGAATATCCTCCTTAG-3′ and 5′-GTGTACCGAAGACTGCACTTAAGTTGGCGCGTTAGACTGC
GTGTAGGCTGGAGCTGCTTCG-3′, yielding a 1,097-bp zapC
fragment (chromosomal sequences are underlined), which was recombined with the chromosome of TB10. In CH56, cat
replaces 570 bp of the zapC
gene (from bp −37 to +533), while flanking genes (pyrD
) are intact.
CH57 was obtained by transduction of zapC
from CH56 to TB28, and FLP-mediated eviction of cat
) from CH57 resulted in CH59.
Transduction of zapA<>cat from CH41 to CH59 resulted in CH63.
CH64 was obtained by FLP-mediated eviction of cat from CH41.
CH65, CH66, and CH67 were obtained by transduction of zapB<>aph from LP1 to CH63, CH59, and CH41, respectively.
For LP1, the aph cassette of pKD13 was amplified using 5′-GGTAATCGGGACGAGGATTTTTATCCATCAACGCCTTGCAATTCCGGGGATCCGTCGACC-3′ and 5′-ACACAGTAAAGAAATTACGCGGAAGATGAAGCGTAATCAGTGTAGGCTGGAGCTGCTTCG-3′, yielding a 1,383-bp zapB<>aph fragment that was recombined with the chromosome of TB10. In LP1, aph replaces 251 bp of the zapB (yiiU) gene (from bp −9 to +242).
Strain Slm260/pTB8 (ΔlacIZYA
) was recovered as a solid-blue colony after plating an EZTnKan-2 transposon library of host strain TB43/pTB8 (ΔlacIZYA
) at 30°C on LB agar supplemented with IPTG and 5-bromo-4-chloro-3-indolyl-β-d
-galactopyranoside (X-Gal) (LB-IX), as described previously (9
). Restreaking of the colony on LB-IX gave rise to both solid-blue (Min+
) and solid-white (Min−
) colonies, and the latter were still about half the size of the former. This indicated that, relative to previously described slm
), the zapCSlm260
::EZTnKan-2) lesion conferred a rather subtle growth defect on Min−
cells. The exact site of EZTnKan-2 insertion was determined as was done previously (8
WM3004 was obtained by transduction of zapC<>aph from JW5125 to TX3772.
Cells were routinely grown at 30°C in LB (0.5% NaCl) or M9 minimal medium supplemented with 0.2% maltose, 0.2% Casamino Acids, and 50 μM thiamine (M9-mal). When appropriate, medium was supplemented with 15 (for strains with bla integrated into the chromosome) or 50 μg/ml ampicillin (Amp), 25 μg/ml kanamycin (Kan), 50 μg/ml spectinomycin (Spec), or 25 μg/ml chloramphenicol (Cam). Other details are specified in the text.
Native (untagged) FtsZ was purified as described previously (39
). Untagged ZapC and ZapC(L22P) were purified using a SUMO fusion system (7
). Strain BL21(λDE3)/plysS (Novagen), harboring either pCH322 (PT7
) or pCH372 [PT7
)] was grown overnight in LB-Amp-Cam with 0.1% glucose. The culture was diluted 1:100 into 0.5 liter of LB-Amp-Cam with 0.04% glucose and grown at 37°C to an optical density at 600 nm (OD600
) of 0.5. IPTG was added to 840 μM, and growth was continued at 30°C for 4 h. Cells were harvested by centrifugation (2,600 × g
, 20 min, 4°C), washed once in 20 ml ice-cold 0.9% NaCl, resuspended in 20 ml ice-cold cell lysis (CL) buffer (50 mM NaH2
, 300 mM NaCl, 10 mM imidazole, pH 8.0), flash frozen in dry ice-acetone, and stored at −80°C. Cells were broken by swirling the tube in a 37°C water bath to quickly thaw the suspension, followed by two more quick-freeze-thaw cycles. After addition of 1 μl Benzonase (Novagen), the lysate was incubated on ice for 30 min and, following brief sonication, subjected to centrifugation (175,000 × g
, 90 min, 4°C). The majority of H-SUMO-ZapC or H-SUMO-ZapC(L22P) was present in the supernatant, 2.5-ml portions of which were loaded on 0.5-ml columns of Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen) preequilibrated in CL buffer. Columns were washed with 4 × 1.0 ml of CL buffer containing 20 mM imidazole and 3 × 0.35 ml of CL buffer containing 50 mM imidazole, and bound protein was eluted with 4 × 0.25 ml of CL buffer containing 250 mM imidazole. Peak fractions were pooled and dialyzed against buffer A (50 mM Tris·Cl, 150 mM NaCl, 1 mM dithiothreitol [DTT], 10% glycerol, pH 8.0). The dialysate was brought to 0.2% NP-40, H-UlpI protease was added to a final molar ratio of 1:300 (protease/substrate), and the mixture was incubated overnight on ice. To capture the His-tagged protease and freed H-SUMO tag, the mixture was brought to 10 mM imidazole and loaded on a 0.5-ml Ni-NTA-agarose column equilibrated in buffer A containing 10 mM imidazole. The column was washed with 6 × 0.25 ml of buffer A containing 10 mM imidazole. Peak fractions of the flowthrough and wash, containing tagless ZapC or ZapC(L22P), were pooled and dialyzed against buffer B (50 mM Tris·Cl, 100 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 10% glycerol, pH 8.0) and further fractionated by anion-exchange chromatography on a Mono-Q column (Pharmacia) with a linear 100 to 600 mM NaCl gradient in the same buffer. The majority of ZapC or ZapC(L22P) eluted between 300 and 350 or 350 and 450 mM NaCl, respectively. Peak fractions were pooled, dialyzed into buffer C (20 mM Tris·Cl, 100 mM KCl, 1 mM EDTA, 10% glycerol, pH 8.0), and stored at −80°C.
A calibrated Superose-12 column was equilibrated in buffer (20 mM Tris·Cl, 100 mM KCl, 1 mM EDTA, pH 8.0), loaded with 25 μg of purified ZapC or ZapC(L22P) (100 μl of 11 μM solution), and run at 400 μl/min with equilibration buffer on an Akta purifier UPC-10 system at 4°C. The column was calibrated with blue dextran (2,000 kDa), sweet potato β-amylase (200 kDa), yeast alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), horse heart cytochrome c (12.4 kDa), and bovine lung aprotinin (6.5 kDa).
Reaction mixtures (100 μl, final volume) containing buffer (50 mM HEPES·KOH, 50 mM KCl, 4 mM MgCl2, pH 7.0), bovine serum albumin (BSA) (3 μM), and other proteins as appropriate were assembled on ice. GTP or GDP was added to 1 mM, and after 5 min at room temperature, the mixtures were subjected to high-speed centrifugation (278,835 × g) for 15 min at 25°C in a Beckman TL-100 ultracentrifuge. Pellets were resuspended in 100 μl of buffer, and equal amounts (11 μl/lane) of pellet and supernatant fractions were loaded on SDS-PAGE gels. After electrophoresis, proteins were stained with Coomassie brilliant blue, gels were digitally imaged with a Fluor-S multi-imager (Bio-Rad), and band intensities were measured using accompanying software.
Light scattering assay.
Light scatter (90o angle) was monitored in a Jobin Yvon Horiba FluoroMax-3 fluorimeter using a wavelength of 350 nm and slit widths set at 1.5 nm. Reactions (150 μl, final volume) were kept at 30°C using a water jacket.
Reactions were started by addition of 2 mM [α-32
P]GTP (~37.5 mCi mmol−1
, corresponding to 3 μCi per reaction). The conversion of [α-32
P]GTP to [α-32
P]GDP at the indicated time was determined by quantitative thin-layer chromatography, essentially as described before (17
Reactions (50 μl, final volume) containing 50 mM HEPES·OH (pH = 7.0), 50 mM KCl, 4 mM MgCl2, and proteins as needed were assembled without nucleotide and placed at 30°C for 2 min. GTP or GDP was added to 1 mM, and 5 min later a 10-μl aliquot was applied to a carbon-coated copper grid (300-mesh) that had been pretreated with 10 μl of a Bacitracin solution (7.5 μg/ml in water) for 0.5 min and wicked dry. After 35 s, the grid was wicked dry, treated with 10 μl uranyl acetate (2%) for 45 s, and wicked dry again. Grids were allowed to dry further in air and were then examined with a JEOL 1200 EX transmission electron microscope (TEM) at 80 kV.
Fluorescence and differential interference contrast (DIC) microscopy (7
), immunofluorescence staining of cells with affinity-purified anti-FtsZ antibodies (37
), measurements of cell parameters (6
), Western analyses with anti-green fluorescent protein (GFP) (10
) or anti-FLAG (74
) antibodies, and yeast two-hybrid assays (43
) were performed as described previously.