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RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli

2008; Springer Nature; Volume: 28; Issue: 3 Linguagem: Inglês

10.1038/emboj.2008.264

1460-2075

Felipe O. Bendezú, Cynthia A. Hale, Thomas G. Bernhardt, Piet A. J. de Boer,

Escherichia coli research studies

Article11 December 2008free access RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli Felipe O Bendezú Felipe O Bendezú Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USAPresent address: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Cynthia A Hale Cynthia A Hale Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Thomas G Bernhardt Thomas G Bernhardt Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USAPresent address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Piet A J de Boer Corresponding Author Piet A J de Boer Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Felipe O Bendezú Felipe O Bendezú Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USAPresent address: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Cynthia A Hale Cynthia A Hale Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Thomas G Bernhardt Thomas G Bernhardt Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USAPresent address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Piet A J de Boer Corresponding Author Piet A J de Boer Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Author Information Felipe O Bendezú1, Cynthia A Hale1, Thomas G Bernhardt1 and Piet A J de Boer 1 1Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA *Corresponding author. Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, W213, 10900 Euclid Avenue, Cleveland, OH 44106, USA. Tel.: +1 216 368 1697; Fax: +1 216 368 3055; E-mail: [email protected] The EMBO Journal (2009)28:193-204https://doi.org/10.1038/emboj.2008.264 There is a Have you seen ...? (February 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The bacterial MreB actin cytoskeleton is required for cell shape maintenance in most non-spherical organisms. In rod-shaped cells such as Escherichia coli, it typically assembles along the long axis in a spiral-like configuration just underneath the cytoplasmic membrane. How this configuration is controlled and how it helps dictate cell shape is unclear. In a new genetic screen for cell shape mutants, we identified RodZ (YfgA) as an important transmembrane component of the cytoskeleton. Loss of RodZ leads to misassembly of MreB into non-spiral structures, and a consequent loss of cell shape. A juxta-membrane domain of RodZ is essential to maintain rod shape, whereas other domains on either side of the membrane have critical, but partially redundant, functions. Though one of these domains resembles a DNA-binding motif, our evidence indicates that it is primarily responsible for association of RodZ with the cytoskeleton. Introduction Bacterial MreB actin has been implicated in cell shape maintenance, chromosome segregation and cell polarization events in a variety of rod-shaped species, including the well-studied model organisms Bacillus subtilis, Caulobacter crescentus and Escherichia coli. The shape of most bacterial cells is dictated by the shape of the murein (peptidoglycan) sacculus, in essence a giant and dynamic cell-shaped molecule that surrounds the entire cytoplasmic membrane. How, despite often considerable turgor pressure, non-coccal organisms manage to mould and maintain this molecule in a particular shape is an unsolved and intensely studied issue (for reviews, see Shih and Rothfield, 2006; Cabeen and Jacobs-Wagner, 2007; den Blaauwen et al, 2008). The MreB protein of E. coli is the only known actin in the cell and, as in other species, accumulates just underneath the cytoplasmic membrane in a spiral/banded-like pattern along the long axis of the cell (Jones et al, 2001; van den Ent et al, 2001; Kruse et al, 2003; Shih et al, 2003; Figge et al, 2004; Gitai et al, 2005). Clear evidence for an important role of MreB in cell shape maintenance came from the isolation of spherical E. coli mreB mutants more than 20 years ago (Wachi et al, 1987), well before the protein was recognized as an actin. About that time the four additional proteins that are currently known to be critical in determining cell shape, MreC, MreD, penicillin-binding protein 2 (PBP2) and RodA were identified as well (Tamaki et al, 1980; Wachi et al, 1989). The genes for MreC and MreD reside with mreB in the mreBCD operon, whereas those for PBP2 (MrdA) and RodA (MrdB) reside in the unlinked mrd operon. Although MreB is cytoplasmic, MreC and PBP2 are bitopic and MreD and RodA are polytopic cytoplasmic membrane species. PBP2 is the only murein synthase in E. coli that is specifically required for extension of the cylindrical portion of the sacculus during cell elongation (Spratt, 1975; de Pedro et al, 2001; Vollmer and Bertsche, 2008). RodA is likely needed for proper PBP2 function (Ishino et al, 1986; de Pedro et al, 2001). MreC forms a dimer and is thought to interact with MreB, MreD and several of the high molecular weight murein synthases (PBPs), including PBP2 (Divakaruni et al, 2005, 2007; Dye et al, 2005; Kruse et al, 2005; van den Ent et al, 2006). MreC, MreD and PBP2 accumulate in a spotty or helical manner along the cell envelope in E. coli, B. subtilis and/or C. crescentus (den Blaauwen et al, 2003; Figge et al, 2004; Divakaruni et al, 2005; Dye et al, 2005; Leaver and Errington, 2005). These localization patterns are reminiscent of that of MreB, as well as of the helical patterns of new murein insertion that have been observed along the cylindrical portions of rod-shaped cells. (Daniel and Errington, 2003; Tiyanont et al, 2006; Divakaruni et al, 2007; Varma et al, 2007). Hence, it is proposed that the helical actin fibres function as cytoplasmic tracks for murein synthase and/or hydrolase activities in the periplasm. This would topologically constrain these activities, resulting in helical insertion of new murein and elongation of the cell (Daniel and Errington, 2003; Figge et al, 2004; Carballido-Lopez et al, 2006). The MreB cytoskeleton has also been implicated in chromosome segregation in several organisms (Kruse et al, 2003, 2006; Soufo and Graumann, 2003; Gitai et al, 2005; Srivastava et al, 2007). However, such a role is not evident in all bacteria (Hu et al, 2007), and additional studies have cast doubt on a critical role of MreB in chromosome segregation in B. subtilis and E. coli (Formstone and Errington, 2005; Karczmarek et al, 2007). To help elucidate the assembly and/or functions of the MreB cytoskeleton in bacteria, we sought to identify additional proteins required for maintaining the normal rod shape of E. coli cells. Although mre and mrd mutants of E. coli can propagate as small spheres on poor medium, they succumb to a lethal division defect on rich medium, unless they are supplied with an extra source of the FtsZ cytokinetic protein (Vinella et al, 1993; Kruse et al, 2005; Bendezu and de Boer, 2008). We took advantage of this property in a genetic screen for mutants that require extra FtsZ for good growth on rich medium. This led us to identify a well-conserved bitopic membrane protein of unknown function (YfgA) as a new cell shape protein that we named RodZ. Our evidence shows that RodZ is a component of the MreB cytoskeleton, and is required for its normal spiral-like configuration. Analyses of domain deletion/substitution variants identify a juxta-membrane portion of RodZ as essential to its function, and indicate that other domains engage the cell shape machinery in distinct ways. Curiously, although one of these domains resembles a DNA-binding motif, our evidence indicates that it has a dominant function in the association of RodZ with the MreB cytoskeleton. Results RodZ (YfgA) is a new cell shape factor in E. coli In E. coli, the five known cell shape maintenance proteins MreBCD and MrdAB are all conditionally essential for growth. Cells lacking any of these proteins propagate stably as small spheres at low mass doubling rates, but form giant non-dividing spheroids at higher ones. An extra supply of the FtsZ division protein, however, suppresses lethality by allowing the shape mutants to propagate as small dividing spheres at higher rates as well (Bendezu and de Boer, 2008). We made use of this latter property in a screen for additional cell shape factors by selecting for transposon mutants that (i) required additional FtsZ for survival or good growth on rich medium and (ii) showed a cell shape defect (see Supplementary data). Mutant strain Rod2352 was especially interesting as it propagated as spheroids and EZTnkan-2 had inserted in yfgA, a gene of unknown function. As mutants both lost rod shape and require an overdose of FtsZ for propagation on LB at or below 30°C (see below), we renamed it rodZ. The gene lies immediately upstream of ispG (gcpE), the product of which catalyses an essential step in isoprenoid biosynthesis (Hecht et al, 2001). We next created ΔrodZ strains (Figure 1A; Supplementary Table S2). Similar to the original insertion mutant, ΔrodZ cells failed to maintain rod shape. The growth properties of ΔrodZ cells were complex, and cell shapes depended to some extent on growth conditions. Compared with the wt parent TB28, the mass doubling time of FB60 [ΔrodZ] in M9-mal minimal medium increased markedly (from ∼64 to >250 min; Tables I and III), but the mutant still formed (small) colonies relatively efficiently on M9 agar (Figure 2A). Cells propagated as spheroids in M9 medium, with few cells displaying more complicated shapes (Figure 2B). Mass doubling time was reduced less in LB medium (∼54 versus ∼35 min at 37°C; Table I). However, although the mutant still formed colonies on LB agar at 42°C and 37°C, it failed to do so at or below 30°C (Figure 2A, right panels). Cell shapes in LB at these lower temperatures were the most complex, with many cells growing into very large non-dividing spheroids bearing one or more cone-like protrusions (Figure 2B). Comparatively, cell shapes were the least abnormal on LB at 42°C, although virtually all cells were still misshapen, with many resembling ellipsoids, lemons and/or very wide rods (Figure 2B). Figure 1.The E. coli rodZ locus and predicted RodZ domain structure. (A) Insertion sites of the rod2352 EZ::Tn transposon (open triangle) and of transposons previously recovered in rodZ (Gerdes et al, 2003) (black triangles), the positions of chromosomal deletion replacements and corresponding strain designations, inserts in plasmids used for initial complementation assays (thick lines), and the results of these assays. Portions of the chromosome that were replaced with an aph cassette, an frt scar sequence or a heterologous transcription regulatory cassette (aph araC PBAD) are indicated by brackets and adjacent numbers refer to base pairs replaced, counting from the start of rodZ. A translation stop (TAA) was placed immediately following codon 155 of rodZ in strain FB61. Plasmids carried the indicated inserts downstream of the lac regulatory region. +, capable of correcting RodZ− and/or IspG− phenotypes; −, incapable of correcting phenotype; ND, not done. (B) Predicted domain organization of RodZ. HTH, Cro/CI-type helix-turn-helix motif (green); +++, basic juxta-membrane (JM) domain (purple); TM, transmembrane domain (black); periplasmic (P) domain (grey) with a region rich in prolines and threonines followed by one that may form several β strands. (C) Comparison of the cytoplasmic portion of RodZ with the N terminus of λ repressor. Basic residues are in blue and acidic ones in red. Helices 1–5 of λCI, and corresponding predicted helices in RodZ are boxed. Identical (*) and similar (.) residues are indicated. The JM domain (residues 85–111) is underlined. Download figure Download PowerPoint Figure 2.Growth and shape phenotypes of ΔrodZ cells, and correction by GFP–RodZ. (A) Spot-titre analyses of wt and ΔrodZ cells. Strains FB60 [ΔrodZ] (uneven rows) and its parent TB28 [wt] (even rows) were grown to density overnight (ON) in M9-mal at 37°C. Cultures were diluted in the same to an optical density at 600 nm (OD600) of 2.4 × 10−2 (columns A and E), 10−3 (columns B and F), 10−4 (columns C and G) and 10−5 (columns D and H), and 10 μl aliquots were spotted on M9-mal (left panel) and LB (right panel) agar. The plates were incubated for 24 (LB) or 48 (M9) h at the indicated temperatures. (B) Phenotypes of wt and ΔrodZ cells. Aliquots of the ON cultures used in (A) were diluted to OD600=0.01 in M9-mal or LB and grown to OD600=0.1–0.3 at the indicated temperatures. Cells were fixed and imaged by DIC microscopy. (C) Suppression of ΔrodZ-associated lethality by extra FtsZ. ON cultures of TB28/pDR3 [wt/Plac::ftsZ] (even rows) and FB60/pDR3 [ΔrodZ /Plac::ftsZ] (uneven rows), grown in LB with 50 μM IPTG, were diluted 104 (columns A and D), 105 (columns B and E) and 106 (columns C and F) times in LB, and 10-μl aliquots were spotted on LB plates containing no (columns A–C) or 50 μM (columns D–F) IPTG. (D) Phenotype of ΔrodZ cells producing extra FtsZ. FB60/pDR3 [ΔrodZ/Plac::ftsZ] cells were grown at 30°C in LB with 50 μM IPTG to OD600=0.3. Note the branching, bulges and oddly placed constrictions. (E) Spiral-like localization of functional GFP–RodZ. FB60(iFB273) [ΔrodZ (Plac::gfp-rodZ)] cells were grown at 30°C to OD600=0.4–0.5 in M9-mal with no (1) or 250 μM (2,3) IPTG and imaged live with DIC (1,2) or fluorescence (3) optics. Bars equal 5 μm (B) or 2 μm (D, E). Download figure Download PowerPoint Table 1. Growth and shape of rodZ cells Straina Relevant genotype IPTG (μM) TDb Shapec LB, 37°C TB28 wt 0 35 Rod FB61 rodZ1−155 0 37 Rod FB60 ΔrodZ 0 54 Odd TB28/pFB290 wt/Plac::rodZ 100 38 Rod FB60/pFB290 ΔrodZ/Plac::rodZ 100 35 Rod M9, 37°C TB28 wt 0 64 Rod FB61 rodZ1−155 0 68 Rod FB60 ΔrodZ 0 274 Spheroid TB28(λFB234) wt (Plac::ispG) 250 62 Rod FB60(λFB234) ΔrodZ (Plac::ispG) 250 298 Spheroid TB28/pFB290 wt/Plac::rodZ 100 92 Rod FB60/pFB290 ΔrodZ/Plac::rodZ 100 62 Rod M9, 30oC TB28(iFB273) wt (Plac::gfp-rodZ) 250 68 Rod FB60(iFB273) ΔrodZ (Plac::gfp-rodZ) 250 68 Rod a Cells were grown at 30°C or 37°C in LB or M9-maltose supplemented with IPTG as indicated. b Mass doubling time in minutes. c Rod, normal rods; spheroid, cells mostly spherical; odd, mixture of bizarre shapes. Table 2. RodZ interactions detected by bacterial two-hybrid assays T18–a RodZ aa missing T25–b Uc RodZ MreB MreC MreD PBP2 RodA RodZ None − +++ ++++ ++ + + − ΔHTH 1–82 − + ++ + − − − HTH-M 85–337 − ++ ++++ − − − − ΔJM 85–110 − +++ ++++ ++ + + − T18–b U RodZ MreB MreC MreD PBP2 RodA T25–RodZa None − +++ ++++ ++ − − − a CyaA domain appended to N terminus of full-length RodZ1−337 (RodZ); RodZ83−337 (ΔHTH); RodZ1−84–MalF1−39–RFP (HTH-M) or RodZ1−84–MalF1−16–RodZ111−337 (ΔJM). b CyaA domain unappended (U) or appended to N terminus of full-length protein, except for MreB wherein the domain was inserted to create a sandwich fusion (see text). c Qualitative measure of β-galactosidase production. Colonies of strain BTH101 [cya-99] carrying appropriate plasmid pairs were patched on indicator plates and inspected for colour development after 24, 30 and 36 h. −, white at 36 h; +, white at 24 h, but light colouring afterwards; ++, light colouring at 24 h and medium blue afterwards; +++, medium blue at 24 h and dark blue afterwards; ++++, dark blue at 24 h and afterwards. Table 3. Phenotypes of RodZ deletion/substitution derivatives Integrated construct Fusion: GFP– Domain(s) missing or substituteda FB60 [ΔrodZ]b TB28 [wt]b TD (min)c % rod-liked,e, d,e Other shapesd,f, d,f Locd,g, d,g Locd,g, d,g iFB273 RodZ1−337 None 68 >99 None Sp Sp iYT22 RodZ1−138–RFP P 86 >99 None Sp Sp iFB285 RodZ1−111 TM+P 518 <1 Spheroid C C iFB289 RodZ1−111–MalF17−39–RFP TM+P 82 45 Mix Sp Sp iFB319 RodZ1−84–MalF1−39–RFP JM+TM+P 301 <1 Spheroid Sp Sp iYT27 RodZ83−337 HTH 76 76 Mix M+Sp M+Sp iFB293 MalF2−14-RodZ111−337 HTH+JM 401 <1 Spheroid M M iFB312 RodZ83−138–RFP HTH+P 602 90% of cells were spheroids of different sizes; mix, cells resembled spheroids; lemons, appeared branched, or had other atypical shapes. g Localization pattern of RodZ variant. Sp, spiral-like and/or spotty along membrane; C, throughout cytoplasm; M, evenly along membrane. Both the growth and shape defects of ΔrodZ cells were fully corrected by pFB290 [Plac::rodZ] or other rodZ constructs, but not by pFB234 [Plac::ispG] (Figure 1; Supplementary Figure S1A; Table I, and see below), showing that neither defect was due to polar effects of the rodZ lesion on ispG or other genes. Recovery of the rodZ::EZTnkan-2 allele in our screen suggested that moderate overexpression of ftsZ is sufficient to suppress lethality of rodZ mutants. Accordingly, FB60 [ΔrodZ] cells that carried pDR3 [Plac::ftsZ] efficiently formed (small) colonies on LB agar at 30°C, provided IPTG was included in the medium (Figure 2C). As with Δmre and Δmrd cells, extra FtsZ allowed ΔrodZ cells to propagate as smaller cells without restoring their shape defects. In M9, this resulted in the formation of smaller spheroids (not shown). In LB, under otherwise non-permissive conditions, extra FtsZ gave rise to heterogeneous populations of severely mis-shaped cells that frequently displayed branches, bulges and oddly placed and/or angled constrictions (Figure 2D). We conclude that rodZ is critical in maintaining the rod shape of E. coli. Similar to the mreBCD and mrdAB genes (Bendezu and de Boer, 2008), rodZ is conditionally essential, and RodZ− lethality under non-permissive conditions can be suppressed by an elevated level of FtsZ. Additionally, ΔrodZ cells displayed a strong medium-dependent growth defect that is not observed in Δmre and Δmrd cells (Bendezu and de Boer, 2008). This defect was not suppressed by elevated levels of FtsZ, by the presence of amino-acid mixtures, or by changing the carbon source (data not shown). Though the reason for this growth phenotype is still unclear, it suggests that RodZ, besides cell shape maintenance, has additional significant function(s) in proliferation. RodZ is a conserved, moderately abundant, transmembrane protein with a putative DNA-binding domain RodZ is a type II (N-in) bitopic membrane protein (Newitt et al, 1999) of 337 residues, and is predicted to possess a Cro/CI-type DNA-binding domain near its N terminus (HTH, residues ∼1–84). This is followed by a highly basic juxta-membrane region (JM, ∼85–110, net charge of +8), a transmembrane domain (TM, ∼111–133) and a substantial periplasmic domain (P, ∼134–337) consisting of a region rich in proline and threonine (∼134–253), and a C-terminal domain that may be rich in β-strands (∼254–337) (Figure 1B and C). Database searches suggest that RodZ-related membrane proteins with a cytoplasmic Cro/CI (Xre)-type DNA-binding domain are present in many Gram-negative as well as Gram-positive organisms from most bacterial phyla (see COG1426; Tatusov et al (2003) and data not shown). The function of none of these has yet been established. We raised antisera against the purified protein and estimated its cellular abundance by quantitative immunoblotting. The results indicated that RodZ is present at ∼650 copies per average exponentially growing cell in LB medium and that its cellular concentration in LB and M9 is about equal (results not shown). As RodZ resembles a transmembrane transcription factor, we initially considered the possibility that it might be needed for expression of one or more of the other known shape proteins. The compatible plasmids pFB174 [PBAD::mreBCD] and pTB59 [Plac::mrdAB] can restore rod shape to ΔmreBCD and ΔmrdAB cells, respectively (Bendezu and de Boer, 2008). However, neither plasmid by itself, or in combination, affected the shape defect of ΔrodZ cells, suggesting that this defect was not due to a lack of any of these proteins (Supplementary Figure S1B). In addition, MreB levels in ΔrodZ cells were close to that in wt cells (Supplementary Figure S1C). Together with the finding that the HTH domain is not strictly needed for imposing rod shape per se (see below), these observations render it unlikely that RodZ is required for rod shape as a transcription factor. RodZ localizes in a spiral-type manner along the membrane Strain FB60(iFB273) [ΔrodZ(Plac::gfp-rodZ)] expresses GFP–RodZ in an IPTG-dependent manner from a construct (iFB273) that was integrated at the chromosomal attHK022 site using the CRIM system (Haldimann and Wanner, 2001). Cells of this strain showed the typical RodZ− morphology upon growth in medium without inducer (Figure 2E1). However, cell morphology became indistinguishable from wt cells in the presence of IPTG at 250 μM or higher, showing that GFP–RodZ is fully capable of restoring rod shape. Fluorescence microscopy showed a spiral-like distribution of the fusion along the length of cells (Figure 2E3), reminiscent of that previously seen for the MreB cytoskeleton (Kruse et al, 2003; Shih et al, 2003). RodZ is part of the spiral-like MreB cytoskeleton Obtaining a fully functional fluorescent version of E. coli MreB by appending fluorescent tags at either terminus proved fruitless (not shown). However, we were able to construct a functional sandwich fusion (MreB–RFPSW) by inserting mCherry RFP between helices 6 and 7 of the protein (van den Ent et al, 2001). We then created strains that produce the MreB–RFPSW sandwich under native regulatory control and as the sole actin in the cell. Substitution of mreB with mreB-rfpsw in these strains was verified by PCR and western blot analyses (Figure 3A and C). Cell morphology and growth rates of mreB-rfpsw strains were indistinguishable from wt controls, and MreB–RFPSW was localized in a banded/spiral-like manner along the long axis of cells (Figure 3B and D). Figure 3.Construction of a functional MreB–RFPSW sandwich fusion, and colocalization of MreB and RodZ. (A–D) Construction and analyses of a chromosomally encoded MreB–RFPSW sandwich fusion. (A) The mre loci of wt and mreB-rfpSW strains, illustrating the location of the rfp ORF within that of mreB in strain FB72 and its derivatives, is shown. The annealing sites and orientations of primers A1 and A2 (pair A) and B1 and B2 (pair B) are indicated. These pairs were used to amplify chromosomal DNA of TB28 [wt] (lanes 2 and 3) or FB72/pCX16 [mreB-rfpSW/sdiA] (lanes 4 and 5) by PCR, and the products were analysed by agarose gel electrophoresis. Size standards (in kb) are shown in lane 1. For (B, C), ON cultures of TB28 [wt], FB66 [yhdE cat] and FB76 [mreB-rfpSW yhdE cat] were diluted in LB to OD600=0.01 and grown at 30°C. Mass doubling rates were determined by measuring OD600 at 1-h intervals. At OD600=0.5, aliquots were used for the determination of cell shape parameters or for the preparation of whole-cell extracts. Note that yhdE cat was used as a selectable marker for transduction of the closely linked mreB-rfpSW allele into various strain backgrounds. (B) The cell doubling times and the average cell length and width (n=200) of the three strains are listed. (C) A western blot of the corresponding extracts from strains TB28 (lane 1), FB66 (2) and FB76 (3) is shown. Each lane received 10 μg total protein, and MreB (37.0 kDa, lanes 1 and 2) and MreB–RFPSW (64.3 kDa, lane 3) were detected by using affinity-purified α-MreB antibodies. The positions of 66, 45 and 36 kDa standards are indicated. (D) DIC (D1) and fluorescence (D2) images of FB83 [mreB-rfpSW yhdE frt] cells that were grown to OD600=0.5 in M9-mal are shown. Note the normal rod shape of cells, and the spiral-like distribution of MreB–RFPSW. (E–G) Colocalization of MreB and RodZ. Corresponding DIC (1), RFP (2), GFP (3) and merged fluorescence (4) images are shown. (E) The colocalization of MreB–RFPSW and GFP–RodZ in rod-shaped cells in which these functional fusions are the only MreB and RodZ proteins present are shown. Note the perfect colocalization in the typical spiral-like cytoskeleton. (F, G) Mreb–RFPSW and GFP–RodZ still colocalize in more disorganized patterns at the periphery of spheroids that are completely devoid of MreC and MreD (F), or of MrdA (PBP2) and MrdB (RodA) (G). The focal plane was near the top of cells in (F, G). (H, I) Location of RodZ in the absence of MreB. Corresponding DIC (1), RFP (2) and GFP (3 and 4) fluorescent images are shown. The panels show the distribution of GFP–RodZ in spheroids that either lack all three Mre proteins (H) or MreB specifically (I). The focal plane was near the top (panels 3) or through the interior (panels 4) of the spheroids. Note the even distribution of GFP–RodZ along the cell membrane, including along that of an intra-cytoplasmic vesicle visible in (I) (arrow). Strains used for (E–I) were: FB101(iFB273) [mreB-rfpSW ΔrodZ (Plac::gfp-rodZ)] (E), FB95(iFB273)/pTB63 [mreB-rfpSW ΔmreCD(Plac::gfp-rodZ)/ftsQAZ] (F), FB90(iFB273)/pTB63 [mreB-rfpSW ΔmrdAB(Plac::gfp-rodZ)/ftsQAZ] (G), FB30(λFB237)/pTB63 [ΔmreBCD(Plac::gfp-rodZ)/ftsQAZ] (H) and FB30(λFB237)/pTB63/pFB206 [ΔmreBCD (Plac::gfp-rodZ)/ftsQAZ /PBAD::mreCD] (I). Cells were grown ON in M9-mal with 250 μM IPTG and either no (E–H) or 0.05% (I) arabinose. After dilution to OD600=0.1 in the same medium, growth was continued to OD600=0.4–0.6, and cells were imaged live. Note that under the same conditions, pFB206 directs the production of sufficient MreC and MreD to correct the shape defect of a ΔmreCD strain (not shown). Bar equals 2 μm. Download figure Download PowerPoint We next co-visualized MreB–RFPSW and GFP–RodZ in cells of strain FB101(iFB273) [mreB-rfpsw ΔrodZ (Plac::gfp-rodZ)] in which production of the former is under native control, that of the latter is IPTG dependent, and the fusions are the sole sources of MreB and RodZ. Cells had a completely normal appearance in the presence of inducer (Figure 3E), implying that both fusions were also functional under these conditions. Notably, the two proteins appeared to colocalize perfectly at all stages of the cell cycle and in all cells examined (Figure 3E, and not shown). This suggested that the MreB cytoskeleton might function as a scaffold for proper localization of RodZ. As any of the other cell shape proteins may associate with this scaffold as well (Kruse et al, 2005), we explored the possibility that they mediate the colocalization of RodZ and MreB. To this end, we co-visualized MreB–RFPSW and GFP–RodZ in strains that completely lack MreC and MreD [ΔmreCD], or PBP2 and RodA [ΔmrdAB]. These strains also carried pTB63 [ftsQAZ], allowing the spherical cells to propagate readily (Bendezu and de Boer, 2008). MreB–RFPSW and GFP–RodZ still colocalized perfectly in either strain, even though the proteins co-accumulated in mostly peripheral clusters/foci rather than in obvious spiral-like patterns (Figure 3F and G). To assess whether such clustering of GFP–RodZ depended on MreB, we examined cells of strain FB30(λFB273)/pTB63 [ΔmreBCD(Plac::gfp-rodZ)/ftsQAZ], which lacks all three Mre proteins, as well as in a related strain that lacks MreB specifically. As illustrated in Figure 3H and I, GFP–RodZ distributed evenly along the membrane in spheroids of either strain, showing that MreB indeed directs the cellular location of RodZ. As their colocalization suggested that RodZ and MreB might interact, we assayed for in vivo interactions between RodZ and the other cell shape proteins using the BACTH bacterial two-hybrid system (Table II; Supplementary Figure S2). This system