Changes in the permeability of Escherichia coli during parasitization by Bdellovibrio bacteriovorus

1971 ◽  
Vol 17 (5) ◽  
pp. 689-697 ◽  
Author(s):  
S. F. Crothers ◽  
J. Robinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Host Cell ◽  
Culture Fluid ◽  
Infection Cycle ◽  
Bdellovibrio Bacteriovorus ◽  
E Coli ◽  
Hepes Buffer ◽  
Per Se ◽  
Ph Range

Bdellovibrio bacteriovorus strain 6-5-S completed a typical infection cycle when incubated with E. coli ML 35 (lac i−z+y−) in 0.025 M Hepes buffer, pH 7.8, supplemented with 0.002 M CaCl2∙2H2O. Growth of this strain of B. bacteriovorus was optimal over the range of pH 7.5–8.5. No growth occurred at pH 6.5. The broad pH range may occur because a buffer per se is not required for growth and multiplication. The parasite failed to multiply in a two-membered culture unsupplemented with Ca2+ or Mg2+.Growth and multiplication of B. bacteriovorus in a two-membered culture were assessed by various parameters, including decrease in absorbance at 520 mμ, and release of materials absorbing at 260 mμ, and at 280 mμ. The onset of lysis of the host cell was accompanied by an increase in the release of materials absorbing at 260 mμ and at 280 mμ. The ratio of the absorbance of these materials at 280 and 260 mμ increased at the same time, from which it may be inferred that probably amino acids or proteins were being released.No β-galactosidase could be detected in the culture fluid of the two-membered culture. The infected E. coli cells were more permeable than uninfected cells to o-nitrophenyl-β-D-galactoside, and to the fluorescent dye 8-aniIino-1-naphthalenesulfonic acid.

Relationship between Bdellovibrio bacteriovorus 6-5-S and autoclaved host bacteria

1972 ◽  
Vol 18 (12) ◽  
pp. 1941-1948 ◽  
Author(s):  
S. F. Crothers ◽  
H. B. Fackrell ◽  
J. C. C. Huang ◽  
J. Robinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Rate ◽  
Reducing Sugars ◽  
Culture Fluid ◽  
Amino Sugars ◽  
Buffer Solution ◽  
Bdellovibrio Bacteriovorus ◽  
Initial Concentration ◽  
E Coli

Bdellovibrio bacteriovorus, strain 6-5-S, multiplied in the presence of washed suspensions of Escherichia coli, Spirillum serpens VHL, and Bacillus megaterium which had been autoclaved for 5 min at 121C. No intracellular life cycle was observed. Bdellovibrio bacteriovorus was also able to multiply in an extract from autoclaved E. coli cells after the cells had been removed by centrifugation. Growth of the parasite was dependent upon the addition of Ca2+ and Mg2+ to the buffer solution. The growth rate and yield of B. bacteriovorus on autoclaved cells were not affected by the initial concentration of the parasite. During multiplication of B. bacteriovorus, amino acids, amino sugars, and reducing sugars were released into the culture fluid.

Investigation into the Efficacy of Bdellovibrio bacteriovorus as a Novel Preharvest Intervention To Control Escherichia coli O157:H7 and Salmonella in Cattle Using an In Vitro Model

2015 ◽  
Vol 78 (9) ◽  
pp. 1745-1749 ◽  
Author(s):  
JENNIFER A. PAGE ◽  
BRIAN LUBBERS ◽  
JOSHUA MAHER ◽  
LINDA RITSCH ◽  
SARA E. GRAGG
Keyword(s):  
Escherichia Coli ◽  
In Vitro Model ◽  
Escherichia Coli O157 ◽  
Bdellovibrio Bacteriovorus ◽  
Gram Negative ◽  
E Coli ◽  
Hepes Buffer ◽  
Vitro Model ◽  
Control Samples

Cattle are an important reservoir for the foodborne pathogens Salmonella and Escherichia coli O157:H7; they frequently harbor these microorganisms in their digestive tracts and shed them in their feces. Thus, there is potential for contamination of cattle hides and, subsequently, carcasses. Interventions aimed at reducing or eliminating pathogen shedding preharvest will also reduce the likelihood of beef product contamination by these pathogens. Therefore, this study used an in vitro model to evaluate Bdellovibrio bacteriovorus, a gram-negative microorganism that preys upon other gram-negative microorganisms, as a preharvest intervention to control Salmonella and E. coli O157:H7. Rumen fluid and feces were inoculated with pansusceptible or antimicrobial-resistant strains of one pathogen. Control samples were treated with HEPES buffer, whereas experimental samples were exposed to HEPES buffer plus B. bacteriovorus. Salmonella and E. coli O157:H7 populations were quantified at 0, 24, 48, and 72 h. The most-probable-number (MPN) technique, followed by streaking onto xylose lysine Tergitol 4 agar, was used to determine Salmonella populations, whereas spread plating onto sorbitol MacConkey agar supplemented with cefixime and tellurite was employed to enumerate E. coli O157:H7. B. bacteriovorus reduced pansusceptible Salmonella in cattle feces by 2.02 Log MPN/g (P = 0.0005) and antimicrobial-resistant Salmonella by 3.79 (P < 0.0001) and 2.24 (P = 0.0013) Log MPN/g after 24 and 48 h, respectively, in comparison to control samples. Significant reductions were not observed for E. coli O157:H7 in rumen or feces. These data suggest that further investigation into B. bacteriovorus efficacy as a preharvest intervention to control Salmonella in cattle is warranted.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

2019 ◽  
Vol 8 (1) ◽  
pp. 15-23
Author(s):  
Takashi Nakamura ◽  
Emi Takeda ◽  
Tomoko Kiryu ◽  
Kentaro Mori ◽  
Miyu Ohori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Industrial Production ◽  
Codon Optimization ◽  
Scale Production ◽  
Enzymatic Production ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix ◽  
Natural Amino Acids

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

STUDIES ON THE RELATIONSHIP BETWEEN VIRUS AND HOST CELL

1951 ◽  
Vol 29 (3) ◽  
pp. 144-148
Author(s):  
R. C. French ◽  
S. M. Lesley ◽  
A. F. Graham ◽  
C. E. van Rooyen
Keyword(s):  
Host Cell ◽  
Trichloroacetic Acid ◽  
E Coli ◽  
Breakdown Mechanism ◽  
Previous Infection ◽  
Per Se ◽  
T7 Phage ◽  
The Relationship

Infection of E. coli with T2r, T4r+, T4r, T5, T6r+, or T6r phages induces the formation of a mechanism which extensively degrades P32 labelled T2r+ phage adsorbed to the cell shortly afterwards, about 50% of the P32 being converted to a form soluble in 5% trichloroacetic acid. Each of the above viruses is as effi cient in this respect as a preliminary infection with T2r+ phage itself. Previous infection of the cells with Tl, T3, or T7 phages does not stimulate this mechanism to break down labelled T2r+ virus. When Tl, T3, or T7 phage, then T2r+ phage, and finally P32 labelled T2r+ phage were added to cells, with an interval of several minutes between each addition, the results indicated that adsorption of T2r+ to the cell was not sufficient per se to stimulate the breakdown of labelled phage. Apparently actual infection of the cell by T2r+ virus was required before the breakdown mechanism was induced.

scholarly journals Dual Predation by Bacteriophage and Bdellovibrio bacteriovorus Can Eradicate Escherichia coli Prey in Situations where Single Predation Cannot

2020 ◽  
Vol 202 (6) ◽  
Author(s):  
Laura Hobley ◽  
J. Kimberley Summers ◽  
Rob Till ◽  
David S. Milner ◽  
Robert J. Atterbury ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Prey Availability ◽  
The United States ◽  
The Other ◽  
Bacteriophage Therapy ◽  
Bdellovibrio Bacteriovorus ◽  
Content Type ◽  
E Coli ◽  
Rapid Acquisition ◽  
Predatory Bacteria

ABSTRACT Bacteria are preyed upon by diverse microbial predators, including bacteriophage and predatory bacteria, such as Bdellovibrio bacteriovorus. While bacteriophage are used as antimicrobial therapies in Eastern Europe and are being applied for compassionate use in the United States, predatory bacteria are only just beginning to reveal their potential therapeutic uses. However, predation by either predator type can falter due to different adaptations arising in the prey bacteria. When testing poultry farm wastewater for novel Bdellovibrio isolates on Escherichia coli prey lawns, individual composite plaques were isolated containing both an RTP (rosette-tailed-phage)-like-phage and a B. bacteriovorus strain and showing central prey lysis and halos of extra lysis. Combining the purified phage with a lab strain of B. bacteriovorus HD100 recapitulated haloed plaques and increased killing of the E. coli prey in liquid culture, showing an effective side-by-side action of these predators compared to their actions alone. Using approximate Bayesian computation to select the best fitting from a variety of different mathematical models demonstrated that the experimental data could be explained only by assuming the existence of three prey phenotypes: (i) sensitive to both predators, (ii) genetically resistant to phage only, and (iii) plastic resistant to B. bacteriovorus only. Although each predator reduces prey availability for the other, high phage numbers did not abolish B. bacteriovorus predation, so both predators are competent to coexist and are causing different selective pressures on the bacterial surface while, in tandem, controlling prey bacterial numbers efficiently. This suggests that combinatorial predator therapy could overcome problems of phage resistance. IMPORTANCE With increasing levels of antibiotic resistance, the development of alternative antibacterial therapies is urgently needed. Two potential alternatives are bacteriophage and predatory bacteria. Bacteriophage therapy has been used, but prey/host specificity and the rapid acquisition of bacterial resistance to bacteriophage are practical considerations. Predatory bacteria are of interest due to their broad Gram-negative bacterial prey range and the lack of simple resistance mechanisms. Here, a bacteriophage and a strain of Bdellovibrio bacteriovorus, preyed side by side on a population of E. coli, causing a significantly greater decrease in prey numbers than either alone. Such combinatorial predator therapy may have greater potential than individual predators since prey surface changes selected for by each predator do not protect prey against the other predator.

scholarly journals Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

2006 ◽  
Vol 55 (9) ◽  
pp. 1265-1270 ◽  
Author(s):  
H. Zhu ◽  
C. A. Hart ◽  
D. Sales ◽  
N. B. Roberts
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Gastric Juice ◽  
Bacterial Killing ◽  
E Coli ◽  
Test Organisms ◽  
H Pylori ◽  
Ph Range ◽  
K 12 ◽  
Rough Mutant

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5–7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1×106 E. coli ml−1 and 1×108 H. pylori ml−1) were pre-incubated with test solutions at 37 °C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml−1 at pH 3.5 reduced viable counts by 100 % for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50 % of the control after 20 min incubation in 1 mg pepsin ml−1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

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