Spontaneous Deletion of a 209-Kilobase-Pair Fragment from the Escherichia coli Genome Occurs with Acquisition of Resistance to an Assortment of Infectious Phages

ABSTRACT To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.

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Local genic base composition impacts protein production and cellular fitness

PeerJ ◽

10.7717/peerj.4286 ◽

2018 ◽

Vol 6 ◽

pp. e4286 ◽

Cited By ~ 2

Author(s):

Erik M. Quandt ◽

Charles C. Traverse ◽

Howard Ochman

Keyword(s):

Escherichia Coli ◽

Base Composition ◽

Protein Production ◽

Purifying Selection ◽

Specific Protein ◽

Compositional Variation ◽

Terminal Portion ◽

A Genome ◽

Selection For ◽

Protein Feature

The maintenance of a G + C content that is higher than the mutational input to a genome provides support for the view that selection serves to increase G + C contents in bacteria. Recent experimental evidence fromEscherichia colidemonstrated that selection for increasing G + C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G + C content acts across all sites within a gene or is confined to particular genic regions or nucleotide positions. We systematically altered the G + C contents of the GFP gene and assayed its effects on the fitness of strains harboring each variant. Fitness differences were attributable to the base compositional variation in the terminal portion of the gene, suggesting a connection to the folding of a specific protein feature. Variants containing sequence features that are thought to result in rapid translation, such as low G + C content and high levels of codon adaptation, displayed highly reduced growth rates. Taken together, our results show that purifying selection acting against A and T mutations most likely results from their tendency to increase the rate of translation, which can perturb the dynamics of protein folding.

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A constraint-based modeling approach to reach an improved chemically defined minimal medium for recombinant antiEpEX-scFv production by Escherichia coli

10.22541/au.161864062.20817783/v1 ◽

2021 ◽

Author(s):

Aidin Behravan ◽

atieh hashemi ◽

Sayed-Amir Marashi

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Cell Growth ◽

Recombinant Protein ◽

Minimal Medium ◽

Protein Production ◽

Feeding Strategy ◽

E Coli ◽

A Genome ◽

Constraint Based Modeling

Increasing demand for recombinant therapeutic proteins highlights the necessity of their yield improvement. Culture medium formulation is a popular approach for bioprocess optimization to improve therapeutic protein production. Constraint-based modeling can empower high-precision optimization through information on how media compounds affect metabolism and cell growth. In the current study, a genome-scale metabolic model (GEMM) of Escherichia coli cells was employed to design strategies of minimal medium supplementation for higher antiEpEX-scFv production. Dynamic flux balance analysis of the recombinant E. coli cell model predicted that ammonium was depleted during the process. Based on the simulations, three amino acids (Asn, Gln and Arg) were chosen to be added to the medium to compensate for ammonium depletion. Experimental validation suggested that the addition of these amino acids (one-by-one, or in combinations) can indeed improve cell growth and recombinant protein production. Then, design of experiment was used to optimize the concentrations of amino acids in the growth medium. About two-fold increase in the growth rate and total scFv expression level was observed using this strategy. We conclude that the GEMM-based approach can provide insights into an effective feeding strategy to improve the production of recombinant protein in E. coli.

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Genome-Scale Metabolic Model Based Engineering of Escherichia Coli Enhances Recombinant Single Chain Antibody Fragment Production

10.21203/rs.3.rs-412220/v1 ◽

2021 ◽

Author(s):

Aidin Behravan ◽

Atieh Hashemi ◽

Sayed-Amir Marashi ◽

Hamideh Fouladiha

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Parent Strain ◽

Antibody Fragment ◽

Metabolic Model ◽

Single Chain ◽

Model Based ◽

A Genome ◽

Genome Scale ◽

Engineered Strain

Abstract Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as scFvs. AntiEpEX-scFv is a small antibody fragment receiving considerable attention for the epithelial cell adhesion molecule (EpCAM) targeting. EpCAM is one of the first discovered-cancer-associated biomarkers highly expressed on various types of solid tumors. Hereby, a genome-scale metabolic model guided engineering strategy was proposed to recognize gene targets for improved antiEpEX-scFv production. Flux balance analysis and FVSEOF algorithm identified several potential genetic targets localized in the glucose import system and pentose phosphate pathway that probably guaranteed an improved yield of scFv. Among the targets predicted by the model, glk gene encoding glucokinase was selected to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). Due to metabolic burden, scFv recombinant expression caused a remarkable decrease in the maximum specific growth rate of the transformed strain. By means of overexpressing glk, presumably increasing carbon flux through the PP pathway, the growth capacity of the E. coli recombinant strain was recovered. Moreover, the engineered strain with glk overexpression successfully increased scfv production. The titer of antiEpEX-scFv reached 235.41 ± 9.53 µg/mL (0.428 g/g DCW) in the engineered strain compared with the parent strain (110.236 ± 7.68 µg/mL; 0.202 g/g DCW). So, model-based prediction was experimentally validated. This approach can be considered for the improvement of other recombinant proteins production.

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The Influence of Insulin, ß-Estradiol, Dexamethasone and Thyroid Hormone on the Secretion of Coagulant and Anticoagulant Proteins by HepG2 Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649798 ◽

1995 ◽

Vol 74 (02) ◽

pp. 686-692 ◽

Cited By ~ 11

Author(s):

René W L M Niessen ◽

Birgit A Pfaffendorf ◽

Augueste Sturk ◽

Roy J Lamping ◽

Marianne C L Schaap ◽

...

Keyword(s):

Thyroid Hormone ◽

Protein C ◽

Protein Production ◽

Culture Medium ◽

Protein S ◽

Cell Protein ◽

Hepatoma Cell Line ◽

Factor X ◽

Factor Ii ◽

At Iii

SummaryAs a basis for regulatory studies on the influence of hormones on (anti)coagulant protein production by hepatocytes, we examined the amounts of the plasma proteins antithrombin III (AT III), protein C, protein S, factor II, factor X, fibrinogen, and prealbumin produced by the hepatoma cell line HepG2, into the culture medium, in the absence and presence of insulin, β-estradiol, dexamethasone and thyroid hormone. Without hormones these cells produced large amounts of fibrinogen (2,452 ± 501 ng/mg cell protein), AT III (447 ± 16 ng/mg cell protein) and factor II (464 ± 31 ng/mg cell protein) and only small amounts of protein C (50 ± 7 ng/mg cell protein) and factor X (55 ± 5 ng/mg cell protein). Thyroid hormone had a slight but significant effect on the enrichment in the culture medium of the anticoagulant protein AT III (1.34-fold) but not on protein C (0.96-fold) and protein S (0.91-fold). This hormone also significantly increased the amounts of the coagulant proteins factor II (1.28-fold), factor X (1.45-fold) and fibrinogen (2.17-fold). Insulin had an overall stimulating effect on the amounts of all the proteins that were investigated. Neither dexamethasone nor ß-estradiol administration did substantially change the amounts of these proteins.We conclude that the HepG2 cell is a useful tool to study the hormonal regulation of the production of (anti)coagulant proteins. We studied the overall process of protein production, i.e., the amounts of proteins produced into the culture medium. Detailed studies have to be performed to establish the specific hormonal effects on the underlying processes, e.g., transcription, translation, cellular processing and transport, and secretion.

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Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

Scientific Reports ◽

10.1038/s41598-021-84859-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

David Gonzalez-Perez ◽

James Ratcliffe ◽

Shu Khan Tan ◽

Mary Chen May Wong ◽

Yi Pei Yee ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Secretion ◽

Protein Production ◽

Secretory Protein ◽

Target Protein ◽

Carrier Protein ◽

Signal Peptides ◽

Carrier Proteins ◽

E Coli ◽

Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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

Pathogens ◽

10.3390/pathogens10050549 ◽

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.

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AcrD of Escherichia coli Is an Aminoglycoside Efflux Pump

Journal of Bacteriology ◽

10.1128/jb.182.6.1754-1756.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1754-1756 ◽

Cited By ~ 141

Author(s):

Emiko Y. Rosenberg ◽

Dzwokai Ma ◽

Hiroshi Nikaido

Keyword(s):

Escherichia Coli ◽

Parent Strain ◽

Efflux Pump ◽

Resistance Nodulation Division

ABSTRACT AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the acrD gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and ΔacrD cells accumulated higher levels of [3H]dihydrostreptomycin and [3H]gentamicin than did the parent strain.

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A Genome-Wide Assay Specifies Only GreA as a Transcription Fidelity Factor in Escherichia coli

G3 Genes|Genome|Genetics ◽

10.1534/g3.118.200209 ◽

2018 ◽

Vol 8 (7) ◽

pp. 2257-2264 ◽

Cited By ~ 9

Author(s):

Charles C. Traverse ◽

Howard Ochman

Keyword(s):

Escherichia Coli ◽

Genome Wide ◽

A Genome

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Contribution of dps to Acid Stress Tolerance and Oxidative Stress Tolerance in Escherichia coli O157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3911-3916.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3911-3916 ◽

Cited By ~ 100

Author(s):

Sang Ho Choi ◽

David J. Baumler ◽

Charles W. Kaspar

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Hydrogen Peroxide ◽

Stationary Phase ◽

Stress Tolerance ◽

Parent Strain ◽

Acid Stress ◽

Acid Tolerance ◽

Gastric Fluid ◽

Acid Challenge

ABSTRACT An Escherichia coli O157:H7dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log10-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log10-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log10CFU/ml while the parent strain decreased by approximately 2 log10 CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.

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