scholarly journals Cell Division in Escherichia coli: Role of FtsL Domains in Septal Localization, Function, and Oligomerization

2000 ◽  
Vol 182 (1) ◽  
pp. 116-129 ◽  
Author(s):  
Jean-Marc Ghigo ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Sodium Dodecyl ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Periplasmic Domain ◽  
Membrane Spanning ◽  
Septal Localization ◽  
And Function

ABSTRACT In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either itsHaemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.

scholarly journals Septal Localization of FtsQ, an Essential Cell Division Protein in Escherichia coli

1999 ◽  
Vol 181 (2) ◽  
pp. 521-530 ◽  
Author(s):  
Joseph C. Chen ◽  
David S. Weiss ◽  
Jean-Marc Ghigo ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Single Copy ◽  
Division Site ◽  
Periplasmic Domain ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Septal Localization

ABSTRACT Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQgene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.

scholarly journals Localization and Function of Early Cell Division Proteins in Filamentous Escherichia coli Cells Lacking Phosphatidylethanolamine

1998 ◽  
Vol 180 (16) ◽  
pp. 4252-4257 ◽  
Author(s):  
Eugenia Mileykovskaya ◽  
Qin Sun ◽  
William Margolin ◽  
William Dowhan
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Ring Structure ◽  
Whole Cells ◽  
Nucleation Sites ◽  
Spiral Structures ◽  
Green Fluorescent ◽  
And Function ◽  
Insight Into

ABSTRACT Escherichia coli cells that contain thepss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.

scholarly journals Proteolytic Activity of YibP Protein in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2595-2602 ◽  
Author(s):  
Toshiharu Ichimura ◽  
Mitsuyoshi Yamazoe ◽  
Maki Maeda ◽  
Chieko Wada ◽  
Sota Hiraga
Keyword(s):  
Escherichia Coli ◽  
Mutant Cell ◽  
Sodium Dodecyl ◽  
Coiled Coil ◽  
Middle Part ◽  
Amino Acid Sequences ◽  
Globular Domain ◽  
Sos Induction ◽  
Ethylmethane Sulfonate ◽  
Membrane Spanning

ABSTRACT Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37°C but not at 42°C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42°C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate β-casein.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

scholarly journals The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 236-243 ◽  
Author(s):  
Daisuke Shiomi ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Additional Function ◽  
Ring Assembly ◽  
C Terminus ◽  
Min Proteins ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

scholarly journals Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum of Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6110-6117 ◽  
Author(s):  
André Piette ◽  
Claudine Fraipont ◽  
Tanneke den Blaauwen ◽  
Mirjam E. G. Aarsman ◽  
Soumya Pastoret ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Structural Determinants ◽  
Membrane Anchor ◽  
Interaction Sites ◽  
Division Site ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

scholarly journals Contribution of the FtsQ Transmembrane Segment to Localization to the Cell Division Site

2007 ◽  
Vol 189 (20) ◽  
pp. 7273-7280 ◽  
Author(s):  
Dirk-Jan Scheffers ◽  
Carine Robichon ◽  
Gert Jan Haan ◽  
Tanneke den Blaauwen ◽  
Gregory Koningstein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Central Component ◽  
Transmembrane Segment ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli

1992 ◽  
Vol 174 (23) ◽  
pp. 7717-7728 ◽  
Author(s):  
Luz-Maria Guzman ◽  
James J. Barondess ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Topological Analysis ◽  
Growth Conditions ◽  
Bacterial Cell Division ◽  
Leucine Zippers ◽  
Protein Dimerization ◽  
Membrane Spanning

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL , was detected through characterization of Tn phoA insertions in a plasmid containing this chromosomal region. Tn phoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.

scholarly journals Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane

2008 ◽  
Vol 191 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Martin Krehenbrink ◽  
Frédéric Pecorari ◽  
Anthony P. Pugsley
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Outer Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Sodium Dodecyl ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Ii Secretion System

ABSTRACT The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

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