scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals A Predicted ABC Transporter, FtsEX, Is Needed for Cell Division in Escherichia coli

2004 ◽  
Vol 186 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Kari L. Schmidt ◽  
Nicholas D. Peterson ◽  
Ryan J. Kustusch ◽  
Mark C. Wissel ◽  
Becky Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Abc Transporter ◽  
Luria Bertani ◽  
Wild Type ◽  
E Coli ◽  
Division Site ◽  
Site Localization ◽  
Free Media ◽  
Mass Increase

ABSTRACT FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.

scholarly journals Escherichia coli Minicell Membranes Are Enriched in Cardiolipin

2001 ◽  
Vol 183 (20) ◽  
pp. 6144-6147 ◽  
Author(s):  
Cecile-Marie Koppelman ◽  
Tanneke Den Blaauwen ◽  
Marc C. Duursma ◽  
Ron M. A. Heeren ◽  
Nanne Nanninga
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Cell Division ◽  
Acyl Chain ◽  
Phospholipid Composition ◽  
Wild Type ◽  
Division Site

ABSTRACT The phospholipid composition of Escherichia coliminicells has been studied as a model for the cell division site. Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol. Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity

2005 ◽  
Vol 187 (8) ◽  
pp. 2727-2736 ◽  
Author(s):  
Sambra D. Redick ◽  
Jesse Stricker ◽  
Gina Briscoe ◽  
Harold P. Erickson
Keyword(s):  
Cell Division ◽  
Structural Model ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Bottom Surface ◽  
Gtpase Activity ◽  
Bacterial Cell Division ◽  
Wild Type ◽  
Negative Effects ◽  
A Subunit

ABSTRACT The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 μM, in contrast to the high GTPase activity of wild-type FtsZ above 1 μM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 μM Kd for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.

scholarly journals Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5491-5499 ◽  
Author(s):  
Sun Nyunt Wai ◽  
Marie Westermark ◽  
Jan Oscarsson ◽  
Jana Jass ◽  
Elke Maier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Single Channel ◽  
Dominant Negative ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Negative Feature

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

scholarly journals Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells ofEscherichia coli

1998 ◽  
Vol 180 (23) ◽  
pp. 6107-6116 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Mirjam E. G. Aarsman ◽  
Arend H. J. Kolk ◽  
Miguel Vicente ◽  
Nanne Nanninga
Keyword(s):  
Cell Division ◽  
Cytoplasmic Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Permissive Temperature ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation

1993 ◽  
Vol 13 (9) ◽  
pp. 5725-5737
Author(s):  
M Au-Fliegner ◽  
E Helmer ◽  
J Casanova ◽  
B M Raaka ◽  
H H Samuels
Keyword(s):  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Dominant Negative ◽  
Heptad Repeat ◽  
Specific Response ◽  
Wild Type ◽  
Negative Effects ◽  
Related Factors ◽  
Response Element ◽  
Response Elements

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

scholarly journals Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine

1996 ◽  
Vol 317 (1) ◽  
pp. 267-272 ◽  
Author(s):  
Martin MEWIES ◽  
Leonard C. PACKMAN ◽  
F. Scott MATHEWS ◽  
Nigel S. SCRUTTON
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Post Translational Modification ◽  
Mutant Proteins ◽  
Trimethylamine Dehydrogenase ◽  
Guanidino Group ◽  
Protein Environment ◽  
Mutagenic Changes ◽  
Positively Charged ◽  
Significant Mass

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 53-62 ◽  
Author(s):  
R E Hollingsworth ◽  
R M Ostroff ◽  
M B Klein ◽  
L A Niswander ◽  
R A Sclafani
Keyword(s):  
Protein Kinase ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Mutant Proteins ◽  
Repair Enzymes ◽  
Induced Mutagenesis

Abstract The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

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