Photosensitization of Escherichia coli and Saccharomyces cerevisiae by phenylheptatriyne from Bidens pilosa

1980 ◽  
Vol 26 (6) ◽  
pp. 698-705 ◽  
Author(s):  
Thor Arnason ◽  
Chi-Kit Wat ◽  
Kelsey Downum ◽  
Etsuo Yamamoto ◽  
Elizabeth Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Singlet Oxygen ◽  
Uv Radiation ◽  
Theory Model ◽  
Survival Curves ◽  
Bidens Pilosa ◽  
E Coli ◽  
Test Organisms

The photosensitizing action of 7-phenylhepta-2,4,6-triyne (PHT), a polyacetylenic compound isolated from Bidens pilosa L. (Asteraceae), has been studied using Escherichia coli and Saccharomyces cerevisiae as the test organisms. The survival curves for E. coli treated with PHT and ultraviolet (UV) radiation were obtained and have been interpreted quantitatively on the basis of a target theory model. The number of "targets" in the cell that must be destroyed before cell death occurs was estimated at six, whereas the dose, D0, that reduced the surviving fraction of the population to 1/e of its value was estimated to be 280 J/m2.Survival was enhanced in aerobic conditions as compared with anaerobic conditions, which is strong evidence that PHT does not behave as a photodynamic sensitizer in vivo. This view was confirmed by work with azide (a quencher of singlet oxygen), D2O (which increases the lifetime of singlet oxygen), and superoxide dismutase (which scavenges superoxide radicals). None of these treatments modified the survival curves significantly, indicating that activated species of O2 are probably not involved in photosensitizations with PHT in vivo.Cell respiration was found to be rapidly inhibited by mild treatments of PHT and UV radiation, suggesting that nuclear metabolism is not the primary target of photosensitization as is the case with another group of photosensitizers, the furanocoumarins. The available evidence indicates that PHT is a representative of a new class of phototoxic compounds. A mechanism of action involving production of free radicals is proposed.

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase

1990 ◽  
Vol 10 (4) ◽  
pp. 1633-1641
Author(s):  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Cell Compartment ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Amber Suppressor

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

1990 ◽  
Vol 10 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Cell Compartment ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Amber Suppressor

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

scholarly journals In vitro turnover numbers do not reflect in vivo activities of yeast enzymes

2021 ◽  
Vol 118 (32) ◽  
pp. e2108391118
Author(s):  
Yu Chen ◽  
Jens Nielsen
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Metabolic Model ◽  
Proteomics Data ◽  
Weak Correlation ◽  
E Coli ◽  
Activity Of Enzymes

Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.

scholarly journals RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192 ◽  
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine γ-synthase, E. coli cystathionine β-lyase, and Saccharomyces cerevisiae cystathionine γ-lyase and development of tools for the investigation of their mechanisms and reaction specificity

2009 ◽  
Vol 87 (2) ◽  
pp. 445-457 ◽  
Author(s):  
Ali Farsi ◽  
Pratik H. Lodha ◽  
Jennifer E. Skanes ◽  
Heidi Los ◽  
Navya Kalidindi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Affinity Purification ◽  
Catalytic Efficiency ◽  
Active Site Residues ◽  
E Coli ◽  
Transsulfuration Pathway ◽  
Reaction Specificity

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine γ-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share ∼30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/Kml-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/Kml-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The ΔmetB and ΔmetC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

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 The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Michael A McAlear ◽  
K Michelle Tuffo ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Dna Replication ◽  
Uv Radiation ◽  
Large Subunit ◽  
Restrictive Temperature ◽  
Replication Factor C ◽  
Replication Factor ◽  
Factor C

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

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 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.

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