SERINE AND THREONINE DEAMINASES OF ESCHERICHIA COLI: ACTIVATORS FOR A CELL-FREE ENZYME

ABSTRACT Fatty acid ethyl esters (FAEEs) were produced in this study by the use of an engineered Escherichia coli p(Microdiesel) strain. Four fed-batch pilot scale cultivations were carried out by first using glycerol as sole carbon source for biomass production before glucose and oleic acid were added as carbon sources. Cultivations yielded a cell density of up to 61 ± 3.1 g of cell dry mass (CDM) per liter and a maximal FAEE content of 25.4% ± 1.1% (wt/wt) of CDM.

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Evaluation of Gaseous Chlorine Dioxide as a Sanitizer for Killing Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, and Yeasts and Molds on Fresh and Fresh-Cut Produce

Journal of Food Protection ◽

10.4315/0362-028x-68.6.1176 ◽

2005 ◽

Vol 68 (6) ◽

pp. 1176-1187 ◽

Cited By ~ 112

Author(s):

KAYE V. SY ◽

MELINDA B. MURRAY ◽

M. DAVID HARRISON ◽

LARRY R. BEUCHAT

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Chlorine Dioxide ◽

Foodborne Pathogens ◽

Escherichia Coli O157 ◽

Gaseous Chlorine Dioxide ◽

Sensory Qualities ◽

A Cell ◽

Fresh Cut ◽

Yeasts And Molds

Gaseous chlorine dioxide (ClO2) was evaluated for effectiveness in killing Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on fresh-cut lettuce, cabbage, and carrot and Salmonella, yeasts, and molds on apples, peaches, tomatoes, and onions. Inoculum (100 μl, ca. 6.8 log CFU) containing five serotypes of Salmonella enterica, five strains of E. coli O157:H7, or five strains of L. monocytogenes was deposited on the skin and cut surfaces of fresh-cut vegetables, dried for 30 min at 22°C, held for 20 h at 4°C, and then incubated for 30 min at 22°C before treatment. The skin surfaces of apples, peaches, tomatoes, and onions were inoculated with 100 μl of a cell suspension (ca. 8.0 log CFU) containing five serotypes of Salmonella, and inoculated produce was allowed to dry for 20 to 22 h at 22°C before treatment. Treatment with ClO2 at 4.1 mg/liter significantly (α = 0.05) reduced the population of foodborne pathogens on all produce. Reductions resulting from this treatment were 3.13 to 4.42 log CFU/g for fresh-cut cabbage, 5.15 to 5.88 log CFU/g for fresh-cut carrots, 1.53 to 1.58 log CFU/g for fresh-cut lettuce, 4.21 log CFU per apple, 4.33 log CFU per tomato, 1.94 log CFU per onion, and 3.23 log CFU per peach. The highest reductions in yeast and mold populations resulting from the same treatment were 1.68 log CFU per apple and 2.65 log CFU per peach. Populations of yeasts and molds on tomatoes and onions were not significantly reduced by treatment with 4.1 mg/liter ClO2. Substantial reductions in populations of pathogens on apples, tomatoes, and onions but not peaches or fresh-cut cabbage, carrot, and lettuce were achieved by treatment with gaseous ClO2 without markedly adverse effects on sensory qualities.

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Reassessing Escherichia coli as a cell factory for biofuel production

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2017.02.010 ◽

2017 ◽

Vol 45 ◽

pp. 92-103 ◽

Cited By ~ 28

Author(s):

Chonglong Wang ◽

Brian F Pfleger ◽

Seon-Won Kim

Keyword(s):

Escherichia Coli ◽

Biofuel Production ◽

Cell Factory ◽

A Cell

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The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein.

Genetics ◽

10.1093/genetics/132.2.375 ◽

1992 ◽

Vol 132 (2) ◽

pp. 375-386 ◽

Cited By ~ 1

Author(s):

A Vincent ◽

S W Liebman

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Dna Sequences ◽

Ribosomal Proteins ◽

Amino Acid Sequences ◽

Ribosomal Protein Genes ◽

Significant Homology ◽

A Cell ◽

Functional Equivalent ◽

Ribosomal Protein S13

Abstract The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

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Potential Repurposing of Known Drugs as Potent Bacterial β-Glucuronidase Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112444927 ◽

2012 ◽

Vol 17 (7) ◽

pp. 957-965 ◽

Cited By ~ 18

Author(s):

Syed Ahmad ◽

Mark A. Hughes ◽

Li-An Yeh ◽

John E. Scott

Keyword(s):

Escherichia Coli ◽

Gastrointestinal Tract ◽

Active Metabolite ◽

Enzyme Assay ◽

Intestinal Bacteria ◽

Tricyclic Antidepressant ◽

Gus Activity ◽

Significant Activity ◽

A Cell ◽

Approved Drugs

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration–approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC50 values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC50 = 388 nM) and the antimalarial mefloquine (IC50 = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC50 values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.

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Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli

Microbiology ◽

10.1099/mic.0.26446-0 ◽

2004 ◽

Vol 150 (2) ◽

pp. 437-446 ◽

Cited By ~ 23

Author(s):

Mei-Shiue Kuo ◽

Kuei-Peng Chen ◽

Whi Fin Wu

Keyword(s):

Escherichia Coli ◽

Lon Protease ◽

Specific Substrate ◽

Galactosidase Activity ◽

Wild Type ◽

Reporter Construct ◽

Fusion Construct ◽

In The Wild ◽

A Cell ◽

Cell Division Inhibitor

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for β-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBAD–rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA Red –lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased β-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.

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Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli

Microbiology ◽

10.1099/mic.0.27937-0 ◽

2005 ◽

Vol 151 (7) ◽

pp. 2331-2342 ◽

Cited By ~ 18

Author(s):

Marian Takáč ◽

Angela Witte ◽

Udo Bläsi

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Functional Analysis ◽

Transmembrane Domains ◽

Class I ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Reading Frame ◽

Double Stranded Dna ◽

A Cell

Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin–endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the −1 reading frame at the 3′ end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage λ Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3…. Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.

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