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 degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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 Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

2008 ◽  
Vol 191 (1) ◽  
pp. 333-346 ◽  
Author(s):  
Gouzel Karimova ◽  
Carine Robichon ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Dependent Manner ◽  
E Coli ◽  
Division Site ◽  
Inner Membrane Protein ◽  
Two Hybrid

ABSTRACT Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

Effect of 5-bromouracil on the growth of a thymine auxotroph of Escherichia coli K-12

1971 ◽  
Vol 17 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Roosevelt J. Jones ◽  
Roger R. Hewitt
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Solid Medium ◽  
Cell Number ◽  
Bacteriostatic Effect ◽  
E Coli ◽  
Static Phase ◽  
Two Phases ◽  
K 12 ◽  
Mass Increase

An atypical viability response to 5-bromouracil has been observed in a thymine auxotroph of E. coli K-12. The response occurs in two phases, the first reflecting tolerance to the analogue during continued exponential growth and cell division. The second is a static phase during which viable number remains constant, while cell number and mass increase at a diminishing rate.During the latter phase filamentous cells increase in number and length. Examination of the cloning potential of cells after 10 h of growth in 5-bromouracil indicated that filamentous cells continue extension on solid medium into non-septate coils that are sterile. Other cells, presumably static when plated, readily form microcolonies free of defective members.Observed responses to penicillin, potentially stabilizing media, or added thymine suggest that 5-bromouracil evokes a bimodal response in this strain. The analogue exerts a bacteriostatic effect on some cells which remain viable for several hours. The bacteriocidal effect, presumably on cells continuing growth, interferes with cell division by preventing septation.

scholarly journals Gonococcal MinD Affects Cell Division inNeisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

2001 ◽  
Vol 183 (21) ◽  
pp. 6253-6264 ◽  
Author(s):  
Jason Szeto ◽  
Sandra Ramirez-Arcos ◽  
Claude Raymond ◽  
Leslie D. Hicks ◽  
Cyril M. Kay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Morphological Changes ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
E Coli ◽  
Two Hybrid

ABSTRACT The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae(MinDNg) in this mutant. Hence, MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinDNg is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinCNg and MinDNg from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinCNg or MinDNg alone did not display noticeable morphological changes. These studies suggest that MinDNg is involved in inhibiting gonococcal cell division, likely in conjunction with MinCNg. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinDNg in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinDNg revealed a novel MinDNgself-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinDNg. These results indicate that MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.

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 Murein (Peptidoglycan) Binding Property of the Essential Cell Division Protein FtsN from Escherichia coli

2004 ◽  
Vol 186 (20) ◽  
pp. 6728-6737 ◽  
Author(s):  
Astrid Ursinus ◽  
Fusinita van den Ent ◽  
Sonja Brechtel ◽  
Miguel de Pedro ◽  
Joachim-Volker Höltje ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Disulfide Bridge ◽  
Binding Property ◽  
Wild Type ◽  
E Coli ◽  
Specific Affinity ◽  
Cell Division Protein ◽  
Truncated Variants

ABSTRACT The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.

scholarly journals LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

2009 ◽  
Vol 191 (16) ◽  
pp. 5094-5107 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
Thuy Dinh ◽  
Thomas G. Bernhardt
Keyword(s):  
Escherichia Coli ◽  
Cell Separation ◽  
Daughter Cell ◽  
Cell Lysis ◽  
Wild Type ◽  
Direct Role ◽  
E Coli ◽  
Division Site ◽  
Gradual Loss ◽  
Daughter Cell Separation

ABSTRACT Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM− cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.

scholarly journals Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

2016 ◽  
Vol 198 (11) ◽  
pp. 1683-1693 ◽  
Author(s):  
Elyse J. Roach ◽  
Charles Wroblewski ◽  
Laura Seidel ◽  
Alison M. Berezuk ◽  
Dyanne Brewer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Bacterial Cell ◽  
Site Directed Mutagenesis ◽  
Bacterial Cell Division ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Z Ring

ABSTRACTBacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associatedproteins (Zap) for stability throughout the process of division. InEscherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure ofEscherichia coliZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Usingin vitroFtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments.IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins inE. colithat associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure ofE. coliZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

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