Synthesis of a Collagen Analog in Escherichia Coli Using Recombinant DNA Technology

1989 ◽  
Vol 174 ◽  
Author(s):  
Ina Goldberg ◽  
A. J. Salerno
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Tandem Repeats ◽  
Recombinant Dna ◽  
Cellular Proteins ◽  
Recombinant Dna Technology ◽  
Synthetic Genes ◽  
E Coli ◽  
Bacterial Protease

AbstractA family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the Clal cloning site of the expression vector pJL6. A representative recombinant plasmid, pACI, with an insert of about 340 bp, was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pACI upon heat induction. The in vivo levels of synthetic gene expression obtained showed that the fusion protein was synthesized in E. coli, but was labile compared to other cellular proteins. This degradation could be significantly reduced by the genetic inhibition of a bacterial protease system.

scholarly journals Sensitive Genetic Screen for Protease Activity Based on a Cyclic AMP Signaling Cascade in Escherichia coli

2000 ◽  
Vol 182 (24) ◽  
pp. 7060-7066 ◽  
Author(s):  
Nathalie Dautin ◽  
Gouzel Karimova ◽  
Agnes Ullmann ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Highly Active Antiretroviral Therapy ◽  
Genetic Test ◽  
Genetic System ◽  
Hiv Protease ◽  
E Coli ◽  
Highly Active ◽  
Proteolytic Activities

ABSTRACT We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya+ phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya−. When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya−phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya+. Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya− phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

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 Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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.

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