Use of an Alternative Pathway for the Synthesis of Isoleucine in Escherichia coli Threonine-Producing Strains

2019 ◽  
Vol 35 (4) ◽  
pp. 42-54 ◽  
Author(s):  
T.V. Yuzbashev ◽  
A.S. Fedorov ◽  
D.M. Bubnov ◽  
S.S. Filippova ◽  
F.V. Bondarenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Alternative Pathway ◽  
Inducible Promoter ◽  
Coli Strain ◽  
Ministry Of Education ◽  
E Coli ◽  
Resource Center ◽  
Threonine Production ◽  
Negative Effect ◽  
Regulated Gene Expression

An E. coli strain in which all known pathways of threonine catabolism were inactivated (Δtdh, ΔltaE, ΔAilvA, ΔtdcB, AyiaY) has been constructed. The possibility of an alternative pathway for the isoleucine synthesis by expressing heterologous citramalate synthase from Leptospira interrogans in an E. coli strain carrying the ΔilvA deletion was demonstrated. It was observed that the cimA overexpression has a negative effect on the threonine production. We developed a system for regulated gene expression based on the inducible promoter PLtetO and TetR repressor of the tetracycline operon. A threonine producing strain B-1201 in which the cimA gene is expressed under the control of the regulated promoter was constructed. A correlation of the threonine productivity and the expression level of the cimA gene was shown by culturing the B-1201 strain in fermenter. The optimal inductor content for the maximum threonine accumulation was also determined. Escherichia coli, strain, threonine, citramalate synthase This work was supported by the Ministry of Education and Science of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA. The authors are grateful to Dr. I. V. Manukhov for providing the MG1655Z1strain and pZE21-lux plasmid, and for teaching techniques of bioluminescence measuring.

scholarly journals Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 549
Author(s):  
Julia Ittensohn ◽  
Jacqueline Hemberger ◽  
Hannah Griffiths ◽  
Maren Keller ◽  
Simone Albrecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein C ◽  
Interleukin 1 ◽  
Coli Strain ◽  
Uropathogenic Escherichia Coli ◽  
Reporter Construct ◽  
Regulation Of Expression ◽  
E Coli ◽  
Strain Cft073 ◽  
Myeloid Differentiation Factor

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

scholarly journals Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants

2020 ◽  
Vol 8 (11) ◽  
pp. 1662
Author(s):  
Zachary R. Stromberg ◽  
Rick E. Masonbrink ◽  
Melha Mellata
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Bacterial Colonization ◽  
Coli Strain ◽  
Fold Change ◽  
Initial Contact ◽  
Lon Protease ◽  
E Coli ◽  
Log2 Fold Change ◽  
Colonization Factors

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryo Yoshida ◽  
Hisashi Hemmi
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Membrane Lipids ◽  
Extreme Environments ◽  
Halophilic Archaea ◽  
Coli Strain ◽  
Glycerol Moiety ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix

Abstract Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

scholarly journals Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli

2019 ◽  
Vol 7 (8) ◽  
pp. 258 ◽  
Author(s):  
Patricia Combarros-Fuertes ◽  
Leticia M. Estevinho ◽  
Rita Teixeira-Santos ◽  
Acácio G. Rodrigues ◽  
Cidália Pina-Vaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Membrane Potential ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Coli Strain ◽  
Antibacterial Action ◽  
Manuka Honey ◽  
E Coli ◽  
Action Mechanisms

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

scholarly journals Complete Genome Sequence of Escherichia coli Strain WG5

2018 ◽  
Vol 6 (2) ◽  
Author(s):  
Lejla Imamovic ◽  
Maria-Anna Misiakou ◽  
Eric van der Helm ◽  
Gianni Panagiotou ◽  
Maite Muniesa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Coli Strain ◽  
Somatic Coliphages ◽  
E Coli

ABSTRACT Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain.

scholarly journals Dynamic Mechanisms of the Bactericidal Action of an Al2O3-TiO2-Ag Granular Material on an Escherichia coli Strain

2015 ◽  
Vol 81 (20) ◽  
pp. 7135-7142 ◽  
Author(s):  
Marie-Anne Tartanson ◽  
Laurence Soussan ◽  
Matthieu Rivallin ◽  
Sophie Pecastaings ◽  
Cristian V. Chis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Granular Material ◽  
Morphological Changes ◽  
Membrane Damage ◽  
Coli Strain ◽  
Cell Integrity ◽  
Bactericidal Action ◽  
Intact Cells ◽  
Content Type ◽  
E Coli

ABSTRACTThe bactericidal activity of an Al2O3-TiO2-Ag granular material against anEscherichia colistrain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics onEscherichia coliusing different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al2O3-TiO2-Ag material, 98% ofE. coliisolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeableE. colicells and 1% of intact cells (105genomic units · ml−1) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.

scholarly journals Preparation of gram quantities of a purified R-factor-mediated penicillinase from Escherichia coli strain W3310

1972 ◽  
Vol 130 (1) ◽  
pp. 55-62 ◽  
Author(s):  
J. Melling ◽  
G. K. Scott
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Coli Strain ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
E Coli ◽  
R Factor

Purified penicillinase, in gram quantities, has been prepared from Escherichia coli strain W3310 by using methods developed to handle large amounts of material. The final product had a specific enzyme activity of 3.08 units/μg of protein, which was over twice as high as that reported previously (Datta & Richmond, 1966). The purified enzyme was similar to that from E. coli strain TEM, but different in molecular weight and some other respects. The differences observed may be a result of the greater purity obtained.

Low Variability of Growth Parameters among Six O157:H7 and Non-O157:H7 Escherichia coli Strains

2014 ◽  
Vol 77 (11) ◽  
pp. 1988-1991
Author(s):  
I. FERNÁNDEZ-ESCUDERO ◽  
I. CARO ◽  
J. MATEO ◽  
J. TEJERO ◽  
E. J. QUINTO
Keyword(s):  
Escherichia Coli ◽  
Generation Time ◽  
Growth Parameters ◽  
Brain Heart Infusion ◽  
Lag Time ◽  
Coli Strain ◽  
Time Data ◽  
E Coli ◽  
Confidence Levels ◽  
Coefficients Of Variation

Five Escherichia coli O157:H7 strains and one nonpathogenic E. coli strain were used. All strains were cultured in brain heart infusion broth and were inoculated in 16-well disposable module cassettes of a Bactometer impedance system. Two initial concentrations were obtained in the wells: 1.37 × 103 and 1.36 × 105 CFU/ml. The impedance measurements were monitored for 72 h at 5, 10, or 15°C, 48 h at 20°C, and 24 h at 25, 30, 35, 40, 45, 50 or 55°C. The lag time and the generation time of each culture were calculated from the detection time data. The coefficients of variation between the strains' growth parameters were low (0.009 to 0.105 for generation time and 0.074 to 0.475 for lag time). An F test showed no significant differences between strains at 5 or 1% confidence levels.

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