THE METABOLIC STABILITY OF NUCLEIC ACIDS IN ESCHERICHIA COLI

1956 ◽  
Vol 2 (6) ◽  
pp. 585-597 ◽  
Author(s):  
L. Siminovitch ◽  
A. F. Graham
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Specific Activity ◽  
Inorganic P ◽  
E Coli ◽  
Unequal Exchange ◽  
Phosphorus Precursor ◽  
Kinetics Of ◽  
Rna And Dna

P32 incorporated in ribo- and desoxyribo-nucleic acids during multiplication of Escherichia coli was retained there as nucleotide phosphorus during subsequent generations. There was little or no unequal exchange of P32 between the nucleotides of either nucleic acid during bacterial multiplication. The kinetics of assimilation of inorganic P32 from medium of constant specific activity into the nucleotides of E. coli nucleic acids indicated that phosphorus was incorporated faster into RNA than DNA. E. coli was propagated for two minutes in the presence of inorganic P32 and the rate at which this "two minute" P32 was incorporated into RNA and DNA phosphorus was measured. About 50 to 60 min. was required for both RNA and DNA phosphorus precursor pools to empty. P32 was incorporated into RNA at a faster rate than into DNA. The final ratio RNAP32/DNAP32 was 50% higher than would be expected from the ratio RNAP/DNAP in E. coli. An equal exchange of phosphorus between RNA and DNA does not occur during bacterial multiplication.

Antimicrobial peptide bsn-37 inhibits the growth of Escherichia coli by targeting its cell wall and membrane

2020 ◽  
Vol 10 (8) ◽  
pp. 1260-1264
Author(s):  
Jingyu Fu ◽  
Hao Yang ◽  
Hongliang Wang ◽  
Jun Ke ◽  
Debao Kong
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Nucleic Acids ◽  
Antimicrobial Peptide ◽  
Mechanism Of Action ◽  
E Coli ◽  
Colony Counting ◽  
Kinetics Of ◽  
Growth And Reproduction

To understand the mechanism of action of the antimicrobial peptide bsn-37 on Escherichia coli (E. coli), we investigated its effects on leakage of ultraviolet-absorbing substances, proteins, and nucleic acids from E. coli CVCC1568. The bacteriostatic kinetics of antimicrobial peptides was determined by colony counting. Our study showed that bsn-37 could effectively inhibit the growth and reproduction of E. coli by disrupting its cell wall and membrane.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Bat Red Blood Cells express Nucleic Acid Sensing Receptors and bind RNA and DNA

2022 ◽  
Author(s):  
LK Metthew Lam ◽  
Jane Dobkin ◽  
Kaitlyn A. Eckart ◽  
Ian Gereg ◽  
Andrew DiSalvo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Red Blood Cells ◽  
Immune Function ◽  
Blood Cells ◽  
Toll Like Receptors ◽  
Cpg Dna ◽  
Human Rbcs ◽  
Insight Into ◽  
Rna And Dna

Red blood cells (RBCs) demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. Little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. Here we show that bat RBCs express the nucleic acid-sensing Toll-like receptors TLR7 and TLR9 and bind the nucleic acid ligands, single-stranded RNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens in humans are concealed in bats.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Intrastrand backbone-nucleobase interactions stabilize unwound right-handed helical structures of heteroduplexes of L-aTNA/RNA and SNA/RNA

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yukiko Kamiya ◽  
Tadashi Satoh ◽  
Atsuji Kodama ◽  
Tatsuya Suzuki ◽  
Keiji Murayama ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Crystal Structures ◽  
Prebiotic Chemistry ◽  
Basic Research ◽  
Structural Features ◽  
Helical Structures ◽  
Base Pairs ◽  
Triplex Structure ◽  
Rna And Dna

Abstract Xeno nucleic acids, which are synthetic analogues of natural nucleic acids, have potential for use in nucleic acid drugs and as orthogonal genetic biopolymers and prebiotic precursors. Although few acyclic nucleic acids can stably bind to RNA and DNA, serinol nucleic acid (SNA) and L-threoninol nucleic acid (L-aTNA) stably bind to them. Here we disclose crystal structures of RNA hybridizing with SNA and with L-aTNA. The heteroduplexes show unwound right-handed helical structures. Unlike canonical A-type duplexes, the base pairs in the heteroduplexes align perpendicularly to the helical axes, and consequently helical pitches are large. The unwound helical structures originate from interactions between nucleobases and neighbouring backbones of L-aTNA and SNA through CH–O bonds. In addition, SNA and L-aTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA. The unique structural features of the RNA-recognizing mode of L-aTNA and SNA should prove useful in nanotechnology, biotechnology, and basic research into prebiotic chemistry.

Inactivation of Escherichia coli by Ultrasound Combined with Nisin

2018 ◽  
Vol 81 (6) ◽  
pp. 993-1000 ◽  
Author(s):  
ZUWEN WANG ◽  
XIUFANG BI ◽  
RUI XIANG ◽  
LIYI CHEN ◽  
XIAOPING FENG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synergistic Effect ◽  
Linear Models ◽  
Treatment Time ◽  
Nutrient Broth ◽  
Inactivation Kinetics ◽  
Growth Phases ◽  
E Coli ◽  
Kinetics Of ◽  
Inactivation Effect

ABSTRACT The aim of this study was to investigate the inactivation of nonpathogenic Escherichia coli in nutrient broth and milk through the use of either ultrasound (US) alone or US combined with nisin (US + nisin) treatments. The E. coli cells were treated at 0 to 55°C, 242.04 to 968.16 W/cm2 for 0 to 15 min. The results showed that the inactivation of E. coli by US and US + nisin increased when the temperature, US power density, and treatment time were increased. The inactivation kinetics of E. coli in nutrient broth by US and US + nisin both conformed to linear models. The largest reductions of 2.89 and 2.93 log cycles by US and US + nisin, respectively, were achieved at 968.16 W/cm2 and at 25°C for 15 min. The suspension media of the E. coli cells influenced the inactivation effect of US, while the growth phases of E. coli cells did not affect their resistance to US. Under all experiment conditions of this study, the differences between US and US + nisin in their respective inactivation effects on E. coli were not obvious. The results suggested that nisin had either no effect at all or a weak synergistic effect with US and that the E. coli cells were inactivated mainly by US, thus indicating that the inactivation of E. coli by US is an “all or nothing” event.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

Viability of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Yellow Fat Spreads as Affected by Storage Temperature

2003 ◽  
Vol 66 (4) ◽  
pp. 549-558 ◽  
Author(s):  
SARAH L. HOLLIDAY ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Storage Temperature ◽  
Salt Content ◽  
Inactivation Kinetics ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Inhibition Of Growth ◽  
Time Required ◽  
Kinetics Of

A study was conducted to characterize the survival and inactivation kinetics of a five-serotype mixture of Salmonella (6.23 to 6.55 log10 CFU per 3.5-ml or 4-g sample), a five-strain mixture of Escherichia coli O157:H7 (5.36 to 6.14 log10 CFU per 3.5-ml or 4-g sample), and a six-strain mixture of Listeria monocytogenes (5.91 to 6.18 log10 CFU per 3.5-ml or 4-g sample) inoculated into seven yellow fat spreads (one margarine, one butter-margarine blend, and five dairy and nondairy spreads and toppings) after formulation and processing and stored at 4.4, 10, and 21°C for up to 94 days. Neither Salmonella nor E. coli O157:H7 grew in any of the test products. The time required for the elimination of each pathogen depended on the product and the storage temperature. Death was more rapid at 21°C than at 4.4 or 10°C. Depending on the product, the time required for the elimination of viable cells at 21°C ranged from 5 to 7 days to >94 days for Salmonella, from 3 to 5 days to 28 to 42 days for E. coli O157:H7, and from 10 to 14 days to >94 days for L. monocytogenes. Death was most rapid in a water-continuous spray product (pH 3.66, 4.12% salt) and least rapid in a butter-margarine blend (pH 6.66, 1.88% salt). E. coli O157:H7 died more rapidly than did Salmonella or L. monocytogenes regardless of storage temperature. Salmonella survived longer in high-fat (≥61%) products than in products with lower fat contents. The inhibition of growth is attributed to factors such as acidic pH, salt content, the presence of preservatives, emulsion characteristics, and nutrient deprivation. L. monocytogenes did not grow in six of the test products, but its population increased between 42 and 63 days in a butter-margarine blend stored at 10°C and between 3 and 7 days when the blend was stored at 21°C. On the basis of the experimental parameters examined in this study, traditional margarine and spreads not containing butter are not “potentially hazardous foods” in that they do not support the growth of Salmonella, E. coli O157:H7, or L. monocytogenes.

scholarly journals The degradation of nucleic acids in, and the removal of breakdown products from the small intestines of steers

1980 ◽  
Vol 44 (1) ◽  
pp. 99-112 ◽  
Author(s):  
A. B. McAllan
Keyword(s):  
Uric Acid ◽  
Small Intestine ◽  
Polyethylene Glycol ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Terminal Ileum ◽  
Pyrimidine Nucleosides ◽  
Small Intestines ◽  
Rna And Dna ◽  
Serum And Urine

1. Nucleic acids and breakdown products were estimated in digesta taken from different sites in the small intestines of slaughtered steers given different diets. Amounts passing different sites were compared using cellulose as a non-digestible marker. The validity of this marker was checked with chromic oxide in some experiments. In other experiments, nucleic acids or derivatives were infused into the proximal duodenum of steers receiving diets of approximately equal proportions of flaked maize and hay. The amounts disappearing during passage through the small intestine were estimated using polyethylene glycol (PEG) as a non- absorbable marker.2. In the slaughter experiments the amounts of nucleic acids entering the small intestine varied with the type of diet. RNA and DNA disappeared on average, to extents of 89% and 80% respectively between the abomasum and the terminal ileum, irrespective of the diet. RNA disappearance occurred almost entirely in the proximal quarter of the small intestine, whereas that of DNA extended further along the tract.3. Nucleic acid degradation in the upper small intestine was accompanied by the transient appearance of adenosine, guanosine and pyrimidine nucleosides. These products were in greatest concentration in digesta from the first quarter of the small intestine and had generally completely disappeared by the terminal ileum.4. Of the different substances infused into the small intestine, free nucleic acids were removed to extents greater than 97%, adenine, guanine and uracil had completely disappeared, thymine and xanthine to approximately 80% and 95% and hypoxanthine and cytosine to only 51% and 48% respectively. The nucleosides adenosine and cytidine were also completely removed in the small intestine but were replaced, in part, by the catabolic products inosine plus hypoxanthine or cytosine respectively. Other nucleosides were removed to approximately half the extent of the corresponding bases.5. Serum and urine allantoin and uric acid levels were related to the amounts of purines entering the small intestines in free or bound form.

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