Gamma-irradiation resistant mutants of Escherichia coli: modified enzyme synthesis repressible by arginine and uracil

1969 ◽  
Vol 15 (6) ◽  
pp. 549-554
Author(s):  
Haskel Robern ◽  
F. S. Thatcher
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Feedback Inhibition ◽  
Residual Activity ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Γ Radiation ◽  
Dependent Activity ◽  
In The Wild

The levels of ammonia- or glutamine-dependent carbamyl phosphate synthetase (CPS), aspartate transcarbamylase (ATC), and ornithine transcarbamylase (OTC) were determined in wild-type Escherichia coli and in three mutants resistant to γ-irradiation designated as 1γ, 6γ, and 12γ. Enzyme assays were conducted on the cultures after growth in an arginine–uracil-free medium (AUF) or in AUF medium supplemented with arginine and uracil (C).The growth rate of the wild type and 1γ was the same in AUF or C medium but the levels of CPS, ATC, and OTC in the 1γ mutant ranged from two to four times as high as in the wild-type strain when grown in AUF medium. The 6γ mutant grew more slowly than the wild type and 1γ strains but its rate of growth was the same in all media. When this mutant was grown in AUF medium, the levels of CPS and OTC were as high as in the 1γ mutant but the level of ATC was about the same as in the wild-type strain. The 12γ mutant was the slowest growing and required both arginine and uracil. When we grew the culture in limiting amounts of uracil and excess arginine or vice versa, the ammonia-dependent activity of CPS was higher than in the wild-type strain, but glutamine-dependent CPS, OTC, and ATC were not synthesized by this mutant. Cultures grown in medium C showed only residual activity of all enzymes tested. These results suggest that (1) the 1γ, 6γ, and 12γ mutants have abnormally low pools of arginine and uracil; (2) the mechanism for feedback inhibition is more sensitive to ionizing radiation than the catalytic mechanism; (3) all damage caused by γ-radiation is at the gene level; (4) the locus controlling the synthesis of the ammonia-dependent activity of CPS is the least sensitive to γ-radiation, and is subject to cumulative repression by uracil and arginine.

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 Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in Escherichia coli

2003 ◽  
Vol 185 (11) ◽  
pp. 3469-3472 ◽  
Author(s):  
Joshua D. Tompkins ◽  
Jennifer L. Nelson ◽  
Jill C. Hazel ◽  
Stacy L. Leugers ◽  
Jeffrey D. Stumpf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Adaptive Mutation ◽  
Wild Type ◽  
Pol Iv ◽  
In The Wild ◽  
Dna Polymerase Iv

ABSTRACT The frequencies of nonselected mutations among adaptive Lac+ revertants of Escherichia coli strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.

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 Expression of Hemin Receptor Molecule ChuA Is Influenced by RfaH in Uropathogenic Escherichia coliStrain 536

2001 ◽  
Vol 69 (3) ◽  
pp. 1924-1928 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
Maren Kupfer ◽  
Levente Emödy ◽  
Helge Karch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Receptor Molecule ◽  
E Coli ◽  
Protein Levels ◽  
In The Wild

ABSTRACT The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. colistrain 536. In an rfaH mutant, the chuAtranscript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within thechuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of thechuA determinant and their distribution in E. coli pathogroups are described.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

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.

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