Article Figures & Data
Figures
- FIG. 1.
(A) Effect of salt and temperature on growth of RG60. RG60 (ftsE400::kan) growing in LB with 1% NaCl at 30°C was subcultured into LB with 1% (circles) or no (squares) NaCl at 30°C (open symbols) or 37°C (closed symbols). The inset shows a phase-contrast micrograph of cells harvested at the time indicated by the arrow from the cultures growing with salt. (B) Localization of FtsN. Cells of wild type (MG1655) or RG60 in exponential growth in LB with 1% NaCl at 30°C were fixed, and FtsN was visualized by immunofluorescence microscopy. Arrows point to septal localization of FtsN.
- FIG. 2.
Effect of FtsEX depletion on growth and division. EC1335 (ftsE::kan/pBAD33-ftsEX) was grown in LB with no NaCl but containing either arabinose (closed symbols) or glucose (open symbols) to modulate expression of the plasmid-borne ftsEX genes. Samples were removed periodically to monitor growth by OD600 or cell morphology. The inset shows a phase-contrast micrograph of cells harvested at the time indicated by the arrow.
- FIG. 3.
Localization of FtsE and FtsX to the division site. (A to D) Cells in exponential growth in LB with NaCl were fixed and examined by fluorescence microscopy directly (A and B), by indirect immunofluorescence microscopy (C and D), or by phase-contrast microscopy (C′ and D′). Strains shown are EC1063 (P204-ftsX-gfp) (A); EC1065 (P206-gfp-ftsX) (B); DHB4/pDSW609 (Plac-ftsE-3xHA) (C and D). (E) Relationship between cell length (age) and septal localization of FtsX. 509 cells of EC1063 were measured and scored for the presence (closed symbols) or absence (open symbols) of a fluorescent band at the midcell.
- FIG. 4.
Effect of fts mutations on localization of FtsX to potential division sites in filamentous E. coli cells. Strains induced to express GFP-FtsX or FtsX-GFP were grown under nonpermissive conditions until they became filamentous, at which time they were fixed and examined by fluorescence microscopy to visualize GFP. Relevant division mutations are as follows: ftsZ84(Ts) in EC1158 (A), ftsA12(Ts) in EC1152 (B), and zipA1(Ts) in EC1340 (C); and FtsK depletion in EC1295 (D), FtsQ depletion in EC1179 (E), FtsW depletion in EC1159 (F), and FtsI depletion in EC1181 (G). The arrowhead in panel B points to a faint band sometimes observed in ftsA(Ts) filaments. 4′,6′-Diamidino-2-phenylindole staining was done to verify proper nucleoid segregation (data not shown).
- FIG. 5.
Localization of various division proteins after depletion of FtsEX. The strains used express ftsEX under control of an arabinose-dependent promoter and harbor gfp fusions to different division genes. These strains were grown in parallel in media containing arabinose (short cells) or glucose (filaments) and then fixed and examined by fluorescence microscopy to visualize GFP. (A and B) FtsA-GFP in EC1363; (C and D) ZipA-GFP in EC1391; (E and F) FtsK (1-266)-GFP in EC1386; (G and H) GFP-FtsQ in EC1392; (I and J) GFP-FtsI in EC1366. 4′,6′-Diamidino-2-phenylindole staining was done to verify nucleoid segregation (data not shown).
- FIG. 6.
Model for recruitment of proteins to the septal ring. The first event is polymerization of FtsZ into the Z-ring. FtsA, ZipA, and ZapA bind directly to FtsZ and localize next or concomitantly with Z-ring assembly. Once either FtsA or ZipA has joined the septal ring, the remaining proteins localize in the order indicated. Whether any E. coli proteins are dependent upon ZapA is not yet known.
Tables
- TABLE 1.
Strains and plasmids
Strain or plasmid Relevant features Source or reference Strains MG1655 Wild type Lab collection DHB4 Wild type with F′ lacIq 5 EC549 ftsI::TnphoA (Kanr)/pBAD33-ftsI 38 JOE170 ftsQ::TnphoA (Kanr)/pBAD33-ftsQ 8 JOE563 ftsK::cat/pBAD42-ftsK 7 JMG265 ftsL::TnphoA (Kanr)/pBAD33-ftsL 12 PS223 W3110 zipA1(Ts) 28 RG60 MG1655 ftsE::kan 10 EC1063 MG1655 Δ(attL-lom)::(kan lacIq P204-ftsX-gfp) This study EC1065 MG1655 Δ(attL-lom)::(kan lacIq P206-gfp-ftsX) This study EC1116 MG1655 attφ80::pDSW533 (P206-gfp-ftsX) This study EC1152 EC1116 ftsA12(Ts) leu::Tn10 This study EC1158 EC1116 ftsZ84(Ts) This study EC1159 EC1116 ftsW::kan/pBAD33-ftsW This study EC1179 JOE170 attφ80::pDSW533 (P206-gfp-ftsX) This study EC1180 JMG265 attφ80::pDSW533 (P206-gfp-ftsX) This study EC1181 EC549 attφ80::pDSW533 (P206-gfp-ftsX) This study EC1295 JOE563 Δ(attL-lom)::(kan P204-ftsX-gfp) This study EC1335 MG1655 ftsE::kan/pBAD33-ftsEX This study EC1340 PS223 attφ80::pDSW533 (P206-gfp-ftsX) This study EC1363 EC1335 Δ(attL-lom)::(bla lacIq P206-ftsA-gfp) This study EC1366 EC1335 Δ(attL-lom)::(bla lacIq P204-gfp-ftsI) This study EC1386 EC1335/pCH205 [Plac-ftsK(1-266)-gfp] This study and ref. 18 EC1391 EC1335 Δ(attL-lom)::(bla lacIq P206-zipA-gfp) This study EC1392 EC1335 Δ(attL-lom)::(bla lacIq P206-gfp-ftsQ) This study Plasmids pBAD33 Arabinose regulation (PBAD), p15A ori, Camr 15 pDSW206 P206 promoter, ColE1 ori, Ampr 38 pDSW209 gfp-fusion vector, P206 promoter, ColE1 ori, Ampr 38 pDSW210 gfp-fusion vector, P206 promoter, ColE1 ori, Ampr 38 pDSW533 pJC69-P206-gfp-ftsX (lacIq, oriRR6Kγ, attPφ80, Spcr) This study and ref. 7 pDSW609 pTH18kr-ftsE-3xHA (Plac, Kanr, pSC101 ori) This study and ref. 19 pDSW610 pBAD33-ftsEX This study pDSW621 pBAD33-ftsE This study pDSW622 pBAD33-ftsX This study pDSW636 pDSW209-ftsX This study pDSW637 pDSW210-ftsX This study pDSW638 pDSW206-ftsE-3xHA This study - TABLE 2.
Complementation of the ftsE400::kan allele in RG60a
Plasmid(s) Arabinose Glucose pBAD33 − − pBAD33-ftsE − − pBAD33-ftsX − − pBAD33-ftsEX + − pBAD33-ftsE + pDSW209 (vector) − − pBAD33-ftsE + pDSW636 (gfp-ftsX) + − pBAD33-ftsE + pDSW210 (vector) − − pBAD33-ftsE + pDSW637 (ftsX-gfp) + − pBAD33-ftsX + pDSW206 (vector) − − pBAD33-ftsE + pDSW638 (ftsE-3xHA) + − ↵ a Complementation was determined by colony formation when streaked onto LB plates with no NaCl but containing arabinose or glucose to modulate expression of genes under control of PBAD. IPTG was not necessary for complementation.
- TABLE 3.
Localization of GFP-FtsX or FtsX-GFP in fts mutant backgroundsa
Mutant Growth condition No. of cells scored Avg cell length (μm) Total no. of FtsX rings % Cells with a ring(s) Spacing of ringsb Wild type 30°C 254 4.5 103 44 10 42°C 225 7.2 91 40 18 Temperature-sensitive mutants ftsZ(Ts) 30°C 135 13 36 27 15 42°C 101 34 0 0 >3,400 ftsA(Ts) 30°C 218 6.3 121 56 11 42°C 112 36 95c 60 42 zipA(Ts) 30°C 174 4.0 53 30 13 42°C 105 33 4 3.8 880 Depletion strains FtsK Arabinose 208 3.0 112 54 5.7 Glucose 145 18 261 98 10 FtsQ Arabinose 187 6.9 123 66 11 Glucose 132 28 231 91 18 FtsL Arabinose 220 8.1 157 71 11 Glucose 72 38 152 83 18 FtsW Arabinose 265 4.5 113 43 11 Glucose 61 29 104 89 17 FtsI Arabinose 212 4.5 111 52 8.6 Glucose 111 22 230 92 10 ↵ a Strains used were EC1116, EC1158, EC1152, EC1340, EC1295, EC1179, EC1180, EC1159, and EC1181.
↵ b Units are micrometer per ring. The spacing is a measure of the frequency of rings per unit cell mass and was calculated by dividing the total number of rings (column 5) into the total length of cells or filaments scored (column 3 multiplied by column 4).
↵ c These bands were faint, and so the numbers given understate the degree of dependence on FtsA.
- TABLE 4.
Localization of Fts proteins in FtsEX depletion backgrounda
GFP fusion Sugar No. of cells scored Avg cell length (μm) % of cells with the indicated no. of rings Spacing of ringsb 0 1 2 3 4 5 FtsA-GFP Arabinose 164 4.8 7 85 8 0 0 0 3.6 Glucose 93 35 0 7 23 13 23 34 7.3 ZipA-GFP Arabinose 224 4.1 20 79 1 0 0 0 5.0 Glucose 112 28 4 10 35 19 19 13 9.5 FtsK(1-266)-GFP Arabinose 243 6.3 49 51 0 0 0 0 12 Glucose 101 21 67 29c 4c 0 0 0 55 GFP-FtsQ Arabinose 217 7.2 35 65 0 0 0 0 11 Glucose 131 24 69 27c 4c 0 0 0 67 GFP-FtsI Arabinose 108 5.0 63 37 0 0 0 0 14 Glucose 66 23 68 32c 0 0 0 0 72
