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 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 Zebrafish nephrosin helps host defence against Escherichia coli infection

Open Biology ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 170040 ◽  
Author(s):  
Qianqian Di ◽  
Qing Lin ◽  
Zhibin Huang ◽  
Yali Chi ◽  
Xiaohui Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Innate Immunity ◽  
Host Defence ◽  
Bacterial Burden ◽  
Wild Type ◽  
Lower Survival Rate ◽  
E Coli ◽  
Escherichia Coli Infection ◽  
Effective Models

Neutrophils play important roles in innate immunity and are mainly dependent on various enzyme-containing granules to kill engulfed microorganisms. Zebrafish nephrosin ( npsn ) is specifically expressed in neutrophils; however, its function is largely unknown. Here, we generated an npsn mutant ( npsn smu5 ) via CRISPR/Cas9 to investigate the in vivo function of Npsn. The overall development and number of neutrophils remained unchanged in npsn -deficient mutants, whereas neutrophil antibacterial function was defective. Upon infection with Escherichia coli , the npsn smu5 mutants exhibited a lower survival rate and more severe bacterial burden, as well as augmented inflammatory response to challenge with infection when compared with wild-type embryos, whereas npsn -overexpressing zebrafish exhibited enhanced host defence against E. coli infection. These findings demonstrated that zebrafish Npsn promotes host defence against bacterial infection. Furthermore, our findings suggested that npsn -deficient and -overexpressing zebrafish might serve as effective models of in vivo innate immunity.

scholarly journals In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

2001 ◽  
Vol 183 (7) ◽  
pp. 2259-2264 ◽  
Author(s):  
Yan Wei ◽  
Amy C. Vollmer ◽  
Robert A. LaRossa
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Wild Type ◽  
Potent Inducer ◽  
E Coli ◽  
Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

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 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 A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010 ◽  
Vol 192 (21) ◽  
pp. 5580-5587 ◽  
Author(s):  
Tamara D. James ◽  
Michael Cashel ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Promoter Activation ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

scholarly journals An Escherichia coli Mutant That Makes Exceptionally Long Cells

2015 ◽  
Vol 197 (8) ◽  
pp. 1507-1514 ◽  
Author(s):  
Ziad W. El-Hajj ◽  
Elaine B. Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Luria Broth ◽  
Small Cells ◽  
Content Type ◽  
E Coli ◽  
Coli Mutant ◽  
Rich Media ◽  
Shaped Cell ◽  
Cell Division Protein

ABSTRACTAlthoughEscherichia coliis a very small (1- to 2-μm) rod-shaped cell, here we describe anE. colimutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. Theseextremelyelongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division.IMPORTANCEEscherichia coliis usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usualE. colicell.E. colifilaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects ofE. coliphysiology that are difficult to investigate with small cells.

scholarly journals Protein Synthesis in Escherichia coli with Mischarged tRNA

2003 ◽  
Vol 185 (12) ◽  
pp. 3524-3526 ◽  
Author(s):  
Bokkee Min ◽  
Makoto Kitabatake ◽  
Carla Polycarpo ◽  
Joanne Pelaschier ◽  
Gregory Raczniak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Trna Synthetase ◽  
Wild Type ◽  
Wild Type Level ◽  
E Coli ◽  
Missense Mutant ◽  
Tryptophan Synthetase

ABSTRACT Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNAAsn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNAAsn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNAAsn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

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.

scholarly journals The Stress-Active Cell Division Protein ZapE Alters FtsZ Filament Architecture to Facilitate Division in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Eric C. DiBiasio ◽  
Rebecca A. Dickinson ◽  
Catherine E. Trebino ◽  
Colby N. Ferreira ◽  
Josiah J. Morrison ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
High Temperature ◽  
Environmental Stress ◽  
Atp Hydrolysis ◽  
Major Protein ◽  
Bacterial Cells ◽  
Z Ring ◽  
Cell Division Protein

During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.

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