Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

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 An unusual precursor of 50S ribosomes in a mutant of Escherichia coli

1976 ◽  
Vol 154 (2) ◽  
pp. 311-318 ◽  
Author(s):  
F Markey ◽  
D G Wild
Keyword(s):  
Escherichia Coli ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Coli Strain ◽  
Ribosome Assembly ◽  
Pulse Labelling ◽  
Wild Type ◽  
Inhibition Of Growth ◽  
In The Wild

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein (“47S”) particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

scholarly journals Plasmid DNA Production in Proteome-Reduced Escherichia coli

2020 ◽  
Vol 8 (9) ◽  
pp. 1444
Author(s):  
Mitzi de la Cruz ◽  
Elisa A. Ramírez ◽  
Juan-Carlos Sigala ◽  
José Utrilla ◽  
Alvaro R. Lara
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Novel ◽  
Wild Type ◽  
Batch Mode ◽  
Cell Factories ◽  
Starting Point ◽  
In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

scholarly journals Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis

2020 ◽  
Vol 6 (2) ◽  
pp. 86
Author(s):  
Marina Zoppo ◽  
Fabrizio Fiorentini ◽  
Cosmeri Rizzato ◽  
Mariagrazia Di Luca ◽  
Antonella Lupetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Murine Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Candida Parapsilosis ◽  
Vaginal Candidiasis ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Mutant Strains

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

scholarly journals Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

2016 ◽  
Vol 114 (2) ◽  
pp. E132-E141 ◽  
Author(s):  
Thiago G. P. Alegria ◽  
Diogo A. Meireles ◽  
José R. R. Cussiol ◽  
Martín Hugo ◽  
Madia Trujillo ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Wild Type Strain ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Mutant Strains ◽  
In The Wild ◽  
Acid Hydroperoxide ◽  
Lipoamide Dehydrogenase ◽  
Fatty Acid Hydroperoxides ◽  
Competition Assays

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr fromXylella fastidiosaand were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108M−1⋅s−1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M−1⋅s−1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohrmutant ofPseudomonas aeruginosatoward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohrstrain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohrmutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.

scholarly journals Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of β-Lactamase-Producing Escherichia coli

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Kristin R. Baker ◽  
Helga Høeg Sigurðardóttir ◽  
Bimal Jana ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Lag Phase ◽  
Growth Curve Analysis ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Biogenesis ◽  
Genes Encoding ◽  
In The Wild ◽  
Nonessential Genes

ABSTRACT Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

scholarly journals Inactivation of ibeA and ibeT Results in Decreased Expression of Type 1 Fimbriae in Extraintestinal Pathogenic Escherichia coli Strain BEN2908

2008 ◽  
Vol 76 (9) ◽  
pp. 4129-4136 ◽  
Author(s):  
Mélanie A. M. Cortes ◽  
Julien Gibon ◽  
Nathalie K. Chanteloup ◽  
Maryvonne Moulin-Schouleur ◽  
Philippe Gilot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Coli Strain ◽  
Wild Type ◽  
Adhesive Properties ◽  
Type 1 Fimbriae ◽  
Pathogenic Escherichia Coli ◽  
Genes Encoding

ABSTRACT IbeA in extraintestinal pathogenic Escherichia coli (ExPEC) strains was previously described for its role in invasion. Here we investigated the role of IbeA and IbeT, encoded by a gene located downstream of ibeA, in the adhesion of the avian ExPEC strain BEN2908 to human brain microvascular endothelial cells (HBMEC). The ΔibeA mutant was less adhesive to HBMEC than the wild-type strain BEN2908 was. Because strain BEN2908 also expresses type 1 fimbriae, we measured the adhesion specifically due to IbeA by comparing the adhesive properties of a Δfim derivative of strain BEN2908 to those of a double Δfim ΔibeA mutant. No differences were observed, indicating that the reduction of adhesion in BEN2908 ΔibeA could be due to a decrease in type 1 fimbria expression. We indeed showed that the decreased adhesion of BEN2908 ΔibeA was correlated with a decrease in type 1 fimbria expression. Accordingly, more bacteria had a fim promoter orientated in the off position in a culture of BEN2908 ΔibeA than in a culture of BEN2908. Expression of fimB and fimE, two genes encoding recombinases participating in controlling the orientation of the fim promoter, was decreased in BEN2908 ΔibeA. A reduction of type 1 fimbria expression due to a preferential orientation of the fim promoter in the off position was also seen in an ibeT mutant of strain BEN2908. We finally suggest a role for IbeA and IbeT in modulating the expression of type 1 fimbriae through an as yet unknown mechanism.

scholarly journals Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

2009 ◽  
Vol 191 (17) ◽  
pp. 5538-5548 ◽  
Author(s):  
Pablo I. Nikel ◽  
Jiangfeng Zhu ◽  
Ka-Yiu San ◽  
Beatriz S. Méndez ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Building Blocks ◽  
Flux Analysis ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13C-labeling experiments in chemostat cultures of a wild-type strain, ΔcreB and ΔarcA single mutants, and a ΔcreB ΔarcA double mutant. Continuous cultures were conducted at D = 0.1 h−1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ΔarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ΔarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli

1977 ◽  
Vol 23 (10) ◽  
pp. 1384-1393 ◽  
Author(s):  
Glen D. Armstrong ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Coli Strain ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Isolation And Characterization ◽  
In The Wild ◽  
Resistant Mutants

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolite repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce β-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3′,5′-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.

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