Sensitivity of Escherichia coli to Viral Nucleic Acid, XII. Ca2+-or Ba2+-Facilitated Transfection of Cell Envelope Mutants

1977 ◽  
Vol 32 (5-6) ◽  
pp. 429-433 ◽  
Author(s):  
Akira Taketo
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Nucleoside Transport ◽  
Wild Type ◽  
Viral Nucleic Acid ◽  
E Coli ◽  
The Relationship ◽  
Cellular Competence

Abstract Using various envelope mutants of Escherichia coli, the relationship between cell surface struc­ture and the Ca2+-or Ba2+-dependent competence for transfection was investigated. In contrast with rough strains, smooth bacteria treated with Ca2+ or Ba2+ were incompetent for the trans­fection by ØA RF. In E. coli K12 D21 derivatives, Ca2+-dependent competence remarkably in­ creased by lpsAl mutation and the highest level of competence was attained by further deficiency in glucose units of the LPS. Upon treatment with BaCl2 , strain D21 and its lpsAl mutant became highly competent for ØA RF. The effect of Ba2+ was, however, feeble for lpsAl mutants further deficient in heptose units and/or glucose units. Among different LPS mutants of E. coli B, variation of the Ca2+-or Ba2+-dependent competence was relatively small and even the competence of strain BB12, whose LPS core contained only two KDO units, was nearly equal to that of wild type bacteria. However, the level of cellular competence induced by Ba2+ was not allways parallel to that induced by Ca2+. In mutants deficient in outer membrane protein I, either Ca2+-or Ba2+-dependent competence increased several-fold, whereas in mutants devoid of outer membrane II*, the com­petence decreased considerably. Unlike nucleoside transport, the uptake of DNA was not affected by tsx mutation.

scholarly journals Mechanistic Understanding of the Interactions of Cationic Conjugated Oligo- and Polyelectrolytes with Wild-type and Ampicillin-resistant Escherichia coli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ehsan Zamani ◽  
Shyambo Chatterjee ◽  
Taity Changa ◽  
Cheryl Immethun ◽  
Anandakumar Sarella ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Charge ◽  
Cell Envelope ◽  
Drug Binding ◽  
Outer Cell ◽  
Binding Modes ◽  
Wild Type ◽  
Bacterial Surface ◽  
E Coli ◽  
Future Design

AbstractAn in-depth understanding of cell-drug binding modes and action mechanisms can potentially guide the future design of novel drugs and antimicrobial materials and help to combat antibiotic resistance. Light-harvesting π-conjugated molecules have been demonstrated for their antimicrobial effects, but their impact on bacterial outer cell envelope needs to be studied in detail. Here, we synthesized poly(phenylene) based model cationic conjugated oligo- (2QA-CCOE, 4QA-CCOE) and polyelectrolytes (CCPE), and systematically explored their interactions with the outer cell membrane of wild-type and ampicillin (amp)-resistant Gram-negative bacteria, Escherichia coli (E. coli). Incubation of the E. coli cells in CCOE/CCPE solution inhibited the subsequent bacterial growth in LB media. About 99% growth inhibition was achieved if amp-resistant E. coli was treated for ~3–5 min, 1 h and 6 h with 100 μM of CCPE, 4QA-CCOE, and 2QA-CCOE solutions, respectively. Interestingly, these CCPE and CCOEs inhibited the growth of both wild-type and amp-resistant E. coli to a similar extent. A large surface charge reversal of bacteria upon treatment with CCPE suggested the formation of a coating of CCPE on the outer surface of bacteria; while a low reversal of bacterial surface charge suggested intercalation of CCOEs within the lipid bilayer of bacteria.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli.

1981 ◽  
Vol 77 (2) ◽  
pp. 121-135 ◽  
Author(s):  
H Nikaido ◽  
E Y Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Specific Class ◽  
Wild Type ◽  
Intact Cells ◽  
E Coli ◽  
Low Concentrations ◽  
Transmembrane Pores ◽  
Diffusion Rates ◽  
Type Strains

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.

scholarly journals Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli

2005 ◽  
Vol 187 (6) ◽  
pp. 1913-1922 ◽  
Author(s):  
Anindya S. Ghosh ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Fluorescent Dyes ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
E Coli ◽  
Physiological Processes ◽  
Side Walls

ABSTRACT In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.

scholarly journals The Escherichia coli Efflux Pump TolC Promotes Aggregation of Enteroaggregative E. coli 042

2007 ◽  
Vol 76 (3) ◽  
pp. 1247-1256 ◽  
Author(s):  
Naoko Imuta ◽  
Junichiro Nishi ◽  
Koichi Tokuda ◽  
Rika Fujiyama ◽  
Kunihiro Manago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Microtiter Plate ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Planktonic Cells ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Aggregative Adherence

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.

scholarly journals The Escherichia coli O157 Flagellar Regulatory Gene flhC and Not the Flagellin Gene fliC Impacts Colonization of Cattle

2006 ◽  
Vol 74 (5) ◽  
pp. 2894-2905 ◽  
Author(s):  
Heather S. Dobbin ◽  
Carolyn J. Hovde ◽  
Christopher J. Williams ◽  
Scott A. Minnich
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Oral Dose ◽  
Regulatory Gene ◽  
Wild Type ◽  
E Coli ◽  
Regulatory Mutant ◽  
The Relationship ◽  
Better Than

ABSTRACT A virulent European Escherichia coli O157:H − isolate is nonmotile due to a 12-bp deletion in the flagellar regulatory gene flhC. To investigate the contribution of flhC in the relationship between E. coli O157:H7 and cattle, we constructed a similar flhC regulatory mutant in the well-characterized strain ATCC 43894. There was no difference in the growth rate between the wild type and this regulatory mutant, but phenotypic arrays showed substrate utilization differences. Survival in the bovine gastrointestinal tract and colonization of the rectoanal junction mucosa were assessed. Mixtures of both strains were given orally or rectally to steers or administered into the rumen of cattle dually cannulated at the rumen and duodenum. One day post-oral dose, most rectal/fecal isolates (74%) were the regulatory mutant, but by 3 days post-oral dose and throughout the 42-day experiment, ≥80% of the isolates were wild type. Among steers given a rectal application of both strains, wild-type isolates were the majority of isolates recovered on all days. The regulatory mutant survived better than the wild type in both the rumen and duodenum. To test the role of motility, a filament mutant (ΔfliC) was constructed and similar cattle experiments were performed. On all days post-oral dose, the majority of isolates (64% to 98%) were the filament mutant. In contrast, both strains were recovered equally post-rectal application. Thus, the regulatory mutant survived passage through the bovine gastrointestinal tract better than the wild type but failed to efficiently colonize cattle, and the requirement of flhC for colonization was not dependent on a functional flagellum.

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

1990 ◽  
Vol 36 (8) ◽  
pp. 525-529 ◽  
Author(s):  
Gerardo Naundorf ◽  
Nicholas G. Aumen
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Injury ◽  
Cell Envelope ◽  
Enterobacter Aerogenes ◽  
Ammonia Concentration ◽  
E Coli ◽  
Colony Forming Units ◽  
Pure Cultures ◽  
Cell Envelopes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane. Key words: injury, coliform, ammonia, cell envelope.

scholarly journals Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of “Wild Type” Escherichia coli Strains

2021 ◽  
Vol 12 ◽  
Author(s):  
Sarah A. Blackburn ◽  
Mark Shepherd ◽  
Gary K. Robinson
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Protein Fusion ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
K 12

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA–a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

scholarly journals Role of Sigma E-Regulated Genes in Escherichia coli Uropathogenesis

2006 ◽  
Vol 74 (7) ◽  
pp. 4030-4038 ◽  
Author(s):  
Peter Redford ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Deletion Mutant ◽  
Cell Envelope ◽  
Infection Model ◽  
Wild Type ◽  
E Coli ◽  
Animal Infection ◽  
Strain Cft073 ◽  
Sigma E

ABSTRACT The sigma E regulon encodes proteins for maintenance and repair of the Escherichia coli cell envelope. Previously, we observed that an antirepressor of sigma E, DegS, is essential for uropathogenic E. coli virulence. Here we use a mouse urinary tract infection model to assay the virulence of mutants of E. coli genes described as sigma E dependent. Deletion mutants of candidate genes were made in the uropathogenic E. coli strain CFT073. Swiss Webster female mice were inoculated with a mixture of mutant and wild-type strains. Bladder and kidney homogenates were cultured 2 days after infection, and CFU of the wild type and mutant were compared. Eleven mutants were assayed, and two, CFT073 degP and CFT073 skp, showed significantly diminished survival compared to wild type. DegP is a chaperone and degradase active in the periplasm. Skp is also a periplasmic chaperone. The virulence of the skp deletion mutant could not be restored by complementation with skp. The virulence of the degP deletion mutant, in contrast, could be restored. However, complementation with a degP allele encoding a serine-to-alanine (S210A) mutation at the protease active site fails to restore virulence. Unlike degP mutants in other bacteria, the E. coli degP mutant is tolerant of oxidative stress. It disappears abruptly from bladder and kidney cultures between 6 and 12 hours after inoculation. A mutant of degQ, a close homolog of degP, was not attenuated in mice. This is the first report that the DegP degradase is an E. coli virulence factor in an animal infection model.

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