Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus
2009; Springer Nature; Volume: 29; Issue: 2 Linguagem: Inglês
10.1038/emboj.2009.358
ISSN1460-2075
AutoresJuliane Kühn, Ariane Briegel, Erhard Mörschel, Jörg Kahnt, Katja Leser, Stephanie Wick, Grant J. Jensen, Martin Thanbichler,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle3 December 2009free access Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus Juliane Kühn Juliane Kühn Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Ariane Briegel Ariane Briegel Division of Biology, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Erhard Mörschel Erhard Mörschel Laboratory for Cell Biology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Jörg Kahnt Jörg Kahnt Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Search for more papers by this author Katja Leser Katja Leser Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Stephanie Wick Stephanie Wick Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Grant J Jensen Grant J Jensen Division of Biology, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Martin Thanbichler Corresponding Author Martin Thanbichler Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Juliane Kühn Juliane Kühn Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Ariane Briegel Ariane Briegel Division of Biology, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Erhard Mörschel Erhard Mörschel Laboratory for Cell Biology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Jörg Kahnt Jörg Kahnt Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Search for more papers by this author Katja Leser Katja Leser Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Stephanie Wick Stephanie Wick Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Grant J Jensen Grant J Jensen Division of Biology, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Martin Thanbichler Corresponding Author Martin Thanbichler Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany Search for more papers by this author Author Information Juliane Kühn1,2, Ariane Briegel3,4, Erhard Mörschel5, Jörg Kahnt6, Katja Leser1,2, Stephanie Wick1,2, Grant J Jensen3,4 and Martin Thanbichler 1,2 1Max Planck Institute for Terrestrial Microbiology, Marburg, Germany 2Laboratory for Microbiology, Department of Biology, Philipps University, Marburg, Germany 3Division of Biology, California Institute of Technology, Pasadena, CA, USA 4Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA 5Laboratory for Cell Biology, Department of Biology, Philipps University, Marburg, Germany 6Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany *Corresponding author. Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straβe, 35043 Marburg, Germany. Tel.: +49 6421 178300; Fax: +49 6421 178209; E-mail: [email protected] The EMBO Journal (2010)29:327-339https://doi.org/10.1038/emboj.2009.358 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways. Introduction The cytoskeleton is an integral part of the machinery that controls the temporal and spatial organization of a cell. It is composed of a dynamic network of protein filaments, forming a scaffold that provides structural support to the cell and recruits macromolecular complexes to defined subcellular locations. In recent years, a variety of polymer-forming proteins have been identified in bacteria, acting in cell polarity, morphogenesis, DNA segregation, and cell division. Although many of them are only found in a limited group of organisms, the tubulin homologue FtsZ, the actin homologue MreB, and, to a lesser extent, intermediate filament proteins have emerged to be conserved across a variety of evolutionary lineages (Graumann, 2007; Pogliano, 2008; Thanbichler and Shapiro, 2008). FtsZ polymerizes into a ring-shaped complex at the future division site, acting as a platform for the assembly of the cell division apparatus (Bi and Lutkenhaus, 1991; Goehring and Beckwith, 2005). Dynamic reorganization of this structure is thought to provide a major driving force for cytokinesis (Li et al, 2007; Osawa et al, 2008). Furthermore, the FtsZ ring contributes to normal cell elongation by recruiting the morphogenetic machinery to midcell and thus stimulating medial cell wall growth before the onset of constriction in the α-proteobacterium Caulobacter crescentus (Aaron et al, 2007). In most rod-shaped bacteria, general cell morphology is regulated by an additional cytoskeletal system, based on MreB and its various paralogues. These proteins polymerize into helical cables that line the cytoplasmic membrane and serve to control the spatial distribution of cell wall biosynthetic enzymes, but they have also been implicated in chromosome segregation and cell polarity (Jones et al, 2001; Carballido-Lopez, 2006). A third class of factors involved in bacterial morphogenesis are intermediate filament-like proteins (Izard et al, 1999; Ausmees et al, 2003; Bagchi et al, 2008). The best-characterized member of this class, crescentin, assembles into a polymeric structure at the inner curvature of C. crescentus, mediating the characteristic crescent shape of this bacterium (Ausmees et al, 2003). Unlike MreB, it appears to act mechanically by constraining longitudinal extension of the cell wall, resulting in uneven cell growth and bending (Cabeen et al, 2009). Whereas polymerization of FtsZ and MreB is a highly dynamic process, dependent on the presence of nucleotide cofactors (Mukherjee and Lutkenhaus, 1998; Carballido-Lopez and Errington, 2003; Anderson et al, 2004; Esue et al, 2005), intermediate filament proteins assemble in a spontaneous manner, forming stable filamentous structures both in vitro and in vivo (Ausmees et al, 2003; Bagchi et al, 2008; Charbon et al, 2009). In addition to cytoskeletal filaments, many bacteria contain proteins that polymerize into membrane-associated, lattice-like structures (Stahlberg et al, 2004; Bowman et al, 2008), providing scaffolds for the assembly and localization of other proteins. The first representative of these non-canonical cytoskeletal proteins, DivIVA, was identified in Bacillus subtilis, where it assembles at the cell poles and the division septum and acts as a recruitment factor for the cell division regulator MinCD (Edwards and Errington, 1997). Moreover, during sporulation, it associates with the DNA-binding protein RacA, thereby mediating polar attachment of the chromosomal origin region (Ben-Yehuda et al, 2003; Wu and Errington, 2003). In actinomycetes, DivIVA has adopted a different function, serving as a key regulator of polar growth and development (Flärdh, 2003; Letek et al, 2008). Although DivIVA homologues are restricted to Gram-positive bacteria, C. crescentus and its relatives contain a functionally analogous protein, designated PopZ, which is involved in anchoring of the chromosomal origin regions to the cell poles, polar morphogenesis, and cell polarity (Bowman et al, 2008; Ebersbach et al, 2008). Here, we describe a new class of polymer-forming proteins, designated bactofilins, that are almost universially conserved among bacteria. In C. crescentus, two bactofilins assemble into a membrane-associated laminar structure that shows cell-cycle-dependent polar localization and acts as a platform for the recruitment of a cell wall biosynthetic enzyme involved in polar morphogenesis. Bactofilins display distinct subcellular distributions and dynamics in different bacterial species, suggesting that they are versatile structural elements that have adopted a range of different cellular functions. Results Identification and localization of bactofilin homologues in C. crescentus C. crescentus is characterized by its asymmetric cell division, which gives rise to two morphologically and physiologically distinct daughter cells. The swarmer sibling carries a single polar flagellum and is mobile. The stalked sibling, by constrast, is immobile and displays a long polar protrusion, called the 'stalk', which carries an adhesive organelle at its tip. Whereas the stalked offspring can immediately enter a new round of cell division, swarmer cells first have to differentiate into a stalked cell to continue their cell cycle. To identify factors mediating polar morphogenesis and development in C. crescentus, open reading frames whose transcription is upregulated during the swarmer-to-stalked-cell transition (McGrath et al, 2007) were fused to a gene encoding the yellow fluorescent protein Venus and expressed ectopically under the control of a xylose-inducible promoter (Meisenzahl et al, 1997). Screening the resulting strains for polar fluorescent signals, we identified the thus-far uncharacterized proteins CC1873 and CC3022 (Nierman et al, 2001), now designated bactofilin A (BacA) and bactofilin B (BacB), respectively. BacA has a molecular mass of 16.8 kDa. BacB was originally annotated as a 24.3 kDa protein, but our analyses showed that translation of open reading frame CC3022 initiates at a downstream ATG codon, resulting in a product with a molecular mass of 18.8 kDa (data not shown). BacA and BacB are paralogous proteins (67% sequence similarity) that mainly consist of a conserved domain of unknown function (DUF583), flanked by short, proline-rich terminal regions (Figure 1A; Supplementary Figure S1). Figure 1.Cell-cycle-dependent localization and abundance of BacA and BacB. (A) Schematic representation of BacA and BacB. The position of the conserved DUF583 domain is indicated in green. (B) Localization of BacA and BacB in live cells. Cells of strain JK34 (bacA-ecfp bacB-venus) were grown in PYE-rich medium and visualized by DIC and fluorescence microscopy (bar: 2 μm). (C) Localization of BacA and BacB by immunofluorescence microscopy. Cells of strains CB15N (wild type) and JK5 (ΔbacAB) were probed with anti-BacA and anti-BacB antibodies. Immunocomplexes were detected with a Alexa-Fluor 555-conjugated secondary antibody. To visualize the cells, chromosomal DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). The micrographs shown were created by overlaying the Alexa-Fluor 555 and DAPI signals (bar: 2 μm). (D) Cell-cycle-dependent subcellular localization of BacA and BacB. Swarmer cells of strain JK34 (bacA-ecfp bacB-venus) were transferred onto an agarose pad (t=0 min) and observed as they progressed through the cell cycle, using DIC and fluorescence microscopy (bar: 2 μm). (E) Cell-cycle-dependent abundance of BacA and BacB. Swarmer cells of wild-type strain CB15N were transferred into M2G minimal medium and incubated for the duration of one cell cycle. At the indicated timepoints, samples were taken from the culture and analysed by immunoblotting using anti-BacA, anti-BacB, and anti-CtrA antiserum. The schematic illustrates the morphology of C. crescentus and the subcellular distribution of BacA and BacB at the different cell-cycle stages. Download figure Download PowerPoint To investigate the subcellular localization of BacA and BacB when produced under the control of their native promoters, we constructed a strain (JK34) in which the endogenous bacA and bacB genes were replaced by bacA-ecfp and bacB-venus fusions, respectively. Fluorescent microscopic analysis of JK34 cells showed that the corresponding fusion proteins localized consistently to the stalked pole of the cell (Figure 1B), frequently spreading into the stalk base, whereas no foci were detectable in swarmer cells (data not shown). To verify that the fluorescent tags had no influence on the positioning of BacA and BacB, the localization of the two proteins was further analysed by immunofluorescence microscopy, using affinity purified anti-BacA and anti-BacB antibodies (Figure 1C). In agreement with the in vivo results, both antibodies yielded polar fluorescent signals in wild-type CB15N cells. By contrast, no such signals were detectable in a ΔbacAB double mutant (JK5). As the absence of foci in swarmer cells suggested that BacA and BacB localize dynamically within the cell, time-lapse microscopy was used to follow the subcellular distribution of the two proteins over the course of the cell cycle in strain JK34 (bacA-ecfp bacB-venus). Both bactofilin paralogues were found to accumulate at the stalked pole specifically during the swarmer-to-stalked-cell transition (Figure 1D). The resulting complexes were maintained throughout the subsequent developmental stages and passed on to the stalked progeny on cell division. Earlier work has shown that bacA and bacB mRNA is detectable throughout the cell cycle, although transcription of the two genes peaks during the swarmer-to-stalked-cell transition. To determine whether the absence of bactofilin complexes in swarmer cells is a result of protein degradation, we monitored the cellular abundance of BacA and BacB in wild-type strain CB15N at different developmental stages (Figure 1E). Both proteins were detectable during the swarmer as well as the stalked phase, with their levels remaining constant throughout. Thus, BacA and BacB are present but delocalized in the swarmer progeny and recruited to the stalked pole on transition to the stalked phase. Using quantitative immunoblot analysis, the copy number of BacA and BacB was estimated to about 200 and 20 molecules per cell, respectively (data not shown). BacA and BacB assemble into membrane-associated polymeric sheets As a first approach to determine the function of BacA and BacB, fluorescently labelled derivatives of the two proteins were overproduced in wild-type strain CB15N under the control of a xylose-inducible promoter. Accumulation of the bactofilin homologues was in each case accompanied by distinct morphological changes (Figure 2A). The cells initially became noticeably swollen, with many of them showing unusually high curvature (4 h). Later on, they developed into tightly curled filaments (6 h), which lysed on further incubation. Under these conditions, both fusion proteins formed elongated structures that localized to the inner curvature of the cell. This pattern was strikingly similar to that observed for the intermediate filament-like protein crescentin (CreS) (Ausmees et al, 2003), suggesting that crescentin could act as a scaffold for the assembly of BacA and BacB. However, deletion of creS had no effect on the phenotype induced by overproduction of the bactofilin fusion proteins (Supplementary Figure S2), which indicates that BacA and BacB have an intrinsic propensity to assemble into polymeric complexes. The same morphological defects were also observed on overproduction of the wild-type proteins (data not shown). Figure 2.Assembly of BacA and BacB into membrane-bound polymeric sheets. (A) Filamentous structures and cell shape defects induced by overproduction of BacA and BacB. Cells of wild-type strain CB15N carrying the overexpression plasmid pJK13 (Pxyl-bacA-venus) or pJK14 (Pxyl-bacB-venus), respectively, were grown in PYE-rich medium. Xylose was added to a final concentration of 0.3% to induce synthesis of BacA–Venus or BacB–Venus (t=0 h). At the indicated timepoints, cells were withdrawn from the cultures and visualized by DIC and fluorescence microscopy (bars: 2 μm). The concentrations of BacA–Venus and BacB–Venus were increased about 130- and 360-fold, respectively, over the corresponding wild-type levels (data not shown). (B) Membrane association of BacA and BacB. Whole-cell lysate of wild-type strain CB15N was fractionated by ultracentrifugation. Samples from the lysate, the soluble fraction, and the insoluble membrane fraction were analysed by immunoblotting using anti-BacA, anti-BacB, anti-SpmX, and anti-CtrA antiserum. The intregral membrane protein SpmX (Radhakrishnan et al, 2008) and the cytoplasmic response regulator CtrA (Quon et al, 1996) serve as controls for the fractionation efficiency. (C) Cryo-electron tomography of cells overproducing BacA. Cells of wild-type strain CB15N bearing overexpression plasmid pJK4 (Pxyl-bacA) were grown in PYE medium, induced for 4 h with 0.03% xylose, and analysed by cryo-electron tomography. Shown are a longitudinal section (13-nm slice, panel a) and a cross-section (38-nm slice, panel b) through a reconstructed cell, with the arrow heads pointing to the membrane-associated BacA polymer (bar: 50 nm). Download figure Download PowerPoint Although BacA and BacB are predicted to be soluble, their subcellular localization points to an association with the cell envelope. Cell fractionation studies indeed showed that BacA and the bulk of BacB co-sediment with the cell membranes, even when synthesized at wild-type levels (Figure 2B). Both bactofilin homologues were released in soluble form under alkaline conditions (Supplementary Figure S3), supporting the notion that they are peripheral membrane proteins (Fujiki et al, 1982). To clarify whether BacA and BacB assemble into membrane-bound polymers, the ultrastructure of a strain overproducing BacA was investigated by cryo-electron tomography (Figure 2C). In agreement with the in vivo localization data, all misshaped cells analysed (n=18) showed an extensive sheet-like structure that lined the inner face of the cytoplasmic membrane at a centre-to-centre distance of 9 nm, correlating with the broad bands of fluorescence observed with the tagged proteins. Similar to the fluorescence signals, these polymeric assemblies were consistently localized to the inner cell curvature and generally restricted to regions that displayed unusually strong bending. Analogous structures were observed on overproduction of BacB (Supplementary Figure S4), suggesting that both bactofilin homologues are capable of polymerizing into membrane-associated sheets. It is difficult to acertain whether the polar BacAB clusters formed under normal conditions have a similar architecture, as the surface of the neck region joining the stalk with the cell body is small and highly curved, thus decreasing sensitivity and resolution. However, analysing wild-type cells, we could detect distinct patches of density at the base of the stalk that displayed the same general appearance and distance from the membrane as the sheets observed on bactofilin overproduction (Supplementary Figure S5). This finding supports the hypothesis that BacA and BacB do in fact assemble on the membrane in proximity of the stalked pole. Consistent with these ultrastructural analyses, BacA and BacB spontaneously assembled into filaments on overproduction in Escherichia coli. Both proteins could be isolated in the polymeric state using standard chromatographic methods. Under low-salt conditions, BacA formed large rods and ribbons that were readily visualized by light microscopy (Figure 3A, panel b). Electron microscopic analyses showed that these structures were composed of numerous loosely packed protofilaments, each measuring ∼3 nm in diameter (Figure 3A, panel c). Individual bundles were interconnected by a dense filament network, rending the solution highly viscous. At physiological salt concentrations, the total amount of BacA incorporated into high-molecular weight structures was unchanged (Figure 3B). However, lateral contacts between polymers were reduced, resulting in the accumulation of ribbon-like assemblies comprising two or several protofilaments (Figure 3A, panel d). Polymerization of BacA was still observed at protein concentrations as low as 250 nM (Supplementary Figure S6), indicating that bactofilin monomers interact in a highly efficient manner. Unlike its paralogue, BacB could not be purified in sufficient quantities for detailed biochemical analyses. However, when analysed by electron microscopy, its polymerization behaviour was comparable to that of BacA (Supplementary Figure S7). In addition, we found that BacA and BacB assembled into the same sheet-like structures when overproduced individually in a bactofilin-deficient strain (Supplementary Figure S8), supporting the idea that they share similar biochemical characteristics. Together, our results strongly suggest that the two bactofilin homologues form membrane-associated polymeric clusters, which interfere with normal cell shape when expanding beyond their wild-type dimensions because of overproduction of their constituents. Figure 3.Polymerization of BacA. (A) Polymers formed by BacA. (a) Purified BacA, applied to an 11% SDS–polyacrylamide gel and stained with Coomassie Blue (5 μg of total protein). (b) DIC micrograph of polymers observed after dialysis of BacA (1.6 mg/ml) against 10 mM Tris/HCl (pH 7.5) (bar: 10 μM). (c) Transmission electron micrograph of BacA protofilament bundles formed in a low-salt buffer (buffer LS; see Materials and methods) (bar: 75 nm). (d) Pairs and ribbons of protofilaments obtained by dialysis of BacA against buffer LS containing 300 mM KCl (bar: 50 nm). The same polymerization behaviour was observed in the presence of 100 mM KCl (data not shown). (B) Polymerization efficiency of BacA at different salt concentrations. BacA (0.9 mg/ml) was dialysed against buffer LS containing 0 mM (low salt), 100 mM, or 300 mM KCl. After ultracentrifugation, samples from the supernatant (S) and the pellet (P) were applied to an SDS–gel, and proteins were detected by Coomassie blue staining. Densitometric analysis showed that, in all three conditions, more than 95% of BacA was found in the pellet fraction (data not shown). Download figure Download PowerPoint As the two C. crescentus bactofilin homologues showed similar localization patterns under all conditions tested, it was conceivable that they interacted with each other. To investigate this possibility, we generated strains producing either a BacA–HA (KL7) or a BacB–HA fusion (KL8) in place of the respective wild-type protein. When co-immunoprecipitation analysis was performed on lysates from these strains using anti-HA-affinity beads, BacB co-purified with BacA–HA and vice versa, indicating close association of the two proteins (Figure 4A). In support of this conclusion, a chromosomally encoded BacA–eCFP fusion lost its typical polar localization on overproduction of BacB–Venus, adopting the same filament-like subcellular distribution as its paralogue instead (Figure 4B). Thus, bactofilin sheets appear to represent mixed polymers composed of both BacA and BacB subunits. Figure 4.Assembly of BacA and BacB into co-polymers. (A) Co-immunoprecipitation analysis. HA-tagged derivatives of BacA and BacB were precipitated from cell lysates of strains KL7 (bacA-HA) and KL8 (bacB-HA), respectively, using anti-HA-affinity beads. Proteins co-precipitating with BacA-HA and BacB-HA were probed with anti-BacB and anti-BacA antibodies, respectively. As a control, the same analyses were performed with lysates of wild-type (WT) strain CB15N. (B) Detection of an interaction between BacA and BacB in vivo. Cells of strain MT260 (bacA-ecfp) bearing overexpression plasmid pJK17 (Pxyl-bacB-venus) were grown in PYE-rich medium, induced for 6 h with 0.3% xylose, and visualized by DIC and fluorescence microscopy (bar: 2 μm). Download figure Download PowerPoint Bactofilins mediate polar localization of a peptidoglycan synthase Despite the lack of evolutionary relationship, BacA and BacB resemble the protein localization factors DivIVA from B. subtilis (Edwards et al, 2000; Stahlberg et al, 2004) and PopZ from C. crescentus (Bowman et al, 2008; Ebersbach et al, 2008) in their ability to form polarly localized, membrane-associated polymers. It was, therefore, conceivable that the two bactofilin homologues could have a role in targeting other proteins to the stalked cell pole. To identify possible interaction partners, we generated strains that produced HA-tagged derivatives of BacA or BacB in place of the respective wild-type proteins. After crosslinking with formaldehyde, the hybrid proteins were immunoprecipitated, and co-purifying proteins were identified by mass spectrometry (Figure 5A). Apart from interacting with their respective paralogue, both BacA and BacB associated specifically with one of the five bifunctional penicillin-binding proteins (CC3277) encoded in the C. crescentus genome. CC3277, now designated PbpC (Penicillin-binding protein C), is a 733 amino acid protein, composed of a proline-rich cytoplasmic tail, a single transmembrane helix, and a large periplasmic portion containing a transglycosylase and a transpeptidase domain (Figure 5B). Similar to BacA and BacB, the protein is detectable at equal levels throughout the course of the cell cycle (data not shown). Figure 5.Interaction of BacA and BacB with the penicillin-binding protein PbpC. (A) Identification of proteins interacting with BacA and BacB. Lysates prepared from formaldehyde-treated cells of strains CB15N (wild type), KL7 (bacA-HA), and KL8 (bacB-HA) were subjected to co-immunoprecipitation analysis using anti-HA-affinity beads. Precipitated proteins were resolved in an 11% SDS–polyacrylamide gel and detected by silver staining. Bands specific for the samples from strains KL7 or KL8 were analysed by mass spectrometry. Owing to the limited amount of starting material, only the five most abundant proteins could be identified. (B) Schematic representation of PbpC. The transmembrane helix (residues 86–108) is indicated in green, the transglycosylase domain (TG; residues 133–300) in orange, and the transpeptidase domain (TP; residues 401–668) in blue. (C) Colocalization of BacA and PbpC. Cells of strain JK271 (bacA-ecfp xylX::Pxyl-venus-pbpC) were grown in PYE-rich medium, induced for 1 h with 0.03% xylose, and visualized by DIC and fluorescence microscopy (bar: 2 μm). (D) Detection of an interaction between BacA and PbpC in vivo. Cells of strain MT279 (xylX::Pxyl-venus-pbpC) carrying overexpression plasmid pJK53 (Pxyl-bacA-ecfp) were grown in PYE-rich medium, induced for 4 h with 0.3% xylose, and visualized by DIC and fluorescence microscopy (bar: 2 μm). Note: For strain MT279 lacking an overexpression plasmid, only polar signals were observed when grown under the same conditions (data not shown). (E) Loss of polar PbpC localization on deletion of bacA and bacB. Strains JK308 (ΔpbpC xylX::Pxyl-venus-pbpC) and JK310 (ΔbacAB ΔpbpC xylX::Pxyl-venus-pbpC) were grown in PYE-rich medium, induced for 1 h with 0.03% xylose, and visualized by DIC and fluorescence microscopy (bar: 2 μm). (F) Determination of the PbpC region responsible for interaction with BacA and BacB. Strains JK308 (ΔpbpC xylX::Pxyl-pbpC[AA 1−132]-mCherry) and JK289 (ΔbacAB ΔpbpC xylX::Pxyl-pbpC[AA 1−132]-mCherry) were grown in PYE-rich medium, induced for 2 h with 0.03% xylose, and visualized by DIC and fluorescence microscopy (bar: 2 μm). (G) Reduced stalk length of strains lacking bactofilin homologues and/or PbpC. Strains CB15N (wild type (WT)), MT257 (ΔbacA), MT259 (ΔbacB), JK5 (ΔbacAB), MT304 (ΔpbpC), and JK281 (ΔbacAB ΔpbpC) were grown in PYE-rich medium, diluted 1:20 in minimal medium
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