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 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 PBP1 Is a Component of the Bacillus subtilis Cell Division Machinery

2004 ◽  
Vol 186 (15) ◽  
pp. 5153-5156 ◽  
Author(s):  
Dirk-Jan Scheffers ◽  
Jeffery Errington
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Subtilis Cell ◽  
Knockout Strain ◽  
Green Fluorescent

ABSTRACT Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.

scholarly journals Changes in the Min Oscillation Pattern before and after Cell Birth

2010 ◽  
Vol 192 (16) ◽  
pp. 4134-4142 ◽  
Author(s):  
Jennifer R. Juarez ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
The Other ◽  
Cell Pole ◽  
Daughter Cells ◽  
Division Site ◽  
Before And After ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

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 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 FtsI and FtsW Are Localized to the Septum inEscherichia coli

1998 ◽  
Vol 180 (11) ◽  
pp. 2810-2816 ◽  
Author(s):  
Lilin Wang ◽  
Medhat K. Khattar ◽  
W. D. Donachie ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Specific Inhibitor ◽  
Inescherichia Coli ◽  
Penicillin Binding Protein ◽  
Cell Division Protein

ABSTRACT The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed inftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

scholarly journals Cytological Characterization of YpsB, a Novel Component of the Bacillus subtilis Divisome

2008 ◽  
Vol 190 (21) ◽  
pp. 7096-7107 ◽  
Author(s):  
José Roberto Tavares ◽  
Robson F. de Souza ◽  
Guilherme Louzada Silva Meira ◽  
Frederico J. Gueiros-Filho
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Last Common Ancestor ◽  
Sequence Comparisons ◽  
Gram Positive Bacteria ◽  
Division Site ◽  
Z Ring ◽  
Green Fluorescent

ABSTRACT Cell division in bacteria is carried out by an elaborate molecular machine composed of more than a dozen proteins and known as the divisome. Here we describe the characterization of a new divisome protein in Bacillus subtilis called YpsB. Sequence comparisons and phylogentic analysis demonstrated that YpsB is a paralog of the division site selection protein DivIVA. YpsB is present in several gram-positive bacteria and likely originated from the duplication of a DivIVA-like gene in the last common ancestor of bacteria of the orders Bacillales and Lactobacillales. We used green fluorescent protein microscopy to determine that YpsB localizes to the divisome. Similarly to that for DivIVA, the recruitment of YpsB to the divisome requires late division proteins and occurs significantly after Z-ring formation. In contrast to DivIVA, however, YpsB is not retained at the newly formed cell poles after septation. Deletion analysis suggests that the N terminus of YpsB is required to target the protein to the divisome. The high similarity between the N termini of YpsB and DivIVA suggests that the same region is involved in the targeting of DivIVA. YpsB is not essential for septum formation and does not appear to play a role in septum positioning. However, a ypsB deletion has a synthetic effect when combined with a mutation in the cell division gene ftsA. Thus, we conclude that YpsB is a novel B. subtilis cell division protein whose function has diverged from that of its paralog DivIVA.

scholarly journals Localization of Cell Division Protein FtsK to theEscherichia coli Septum and Identification of a Potential N-Terminal Targeting Domain

1998 ◽  
Vol 180 (5) ◽  
pp. 1296-1304 ◽  
Author(s):  
Xuan-chuan Yu ◽  
Anthony H. Tran ◽  
Qin Sun ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Late Stage ◽  
Fluorescent Protein ◽  
Mutant Protein ◽  
E Coli ◽  
Cell Division Protein ◽  
Green Fluorescent

ABSTRACT Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. colicells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced andftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize inftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.

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