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.

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.

scholarly journals Escherichia coli Physiology in Luria-Bertani Broth

2007 ◽  
Vol 189 (23) ◽  
pp. 8746-8749 ◽  
Author(s):  
Guennadi Sezonov ◽  
Danièle Joseleau-Petit ◽  
Richard D'Ari
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Rate ◽  
Steady State ◽  
Cell Mass ◽  
Carbon Sources ◽  
Luria Bertani ◽  
E Coli ◽  
Luria Bertani Broth ◽  
State Growth

ABSTRACT Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD600) of 7. Surprisingly, however, steady-state growth ceases at an OD600 of 0.3, when the growth rate slows down and cell mass decreases. Growth stops for lack of a utilizable carbon source. The carbon sources for E. coli in Luria-Bertani broth are catabolizable amino acids, not sugars.

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.

Reduction of Escherichia coli O157:H7 Counts on Whole Fresh Apples by Treatment with Sanitizers

2000 ◽  
Vol 63 (6) ◽  
pp. 703-708 ◽  
Author(s):  
MARCY A. WISNIEWSKY ◽  
BONITA A. GLATZ ◽  
MARK L. GLEASON ◽  
CHERYLL A. REITMEIER
Keyword(s):  
Escherichia Coli ◽  
Chlorine Dioxide ◽  
Phosphate Buffer ◽  
Phosphate Buffer Solution ◽  
Escherichia Coli O157 ◽  
Peroxyacetic Acid ◽  
Water Control ◽  
Buffer Solution ◽  
E Coli ◽  
Log Reduction

The objectives of this study were to determine if washing of whole apples with solutions of three different sanitizers (peroxyacetic acid, chlorine dioxide, or a chlorine-phosphate buffer solution) could reduce a contaminating nonpathogenic Escherichia coli O157:H7 population by 5 logs and at what sanitizer concentration and wash time such a reduction could be achieved. Sanitizers were tested at 1, 2, 4, 8, and 16 times the manufacturer's recommended concentration at wash times of 5, 10, and 15 min. Whole, sound Braeburn apples were inoculated with approximately 1 × 108 or 7 × 106 CFU per apple, stored for 24 h, then washed with sterile water (control) or with sanitizers for the prescribed time. Recovered bacteria were enumerated on trypticase soy agar. Washing with water alone reduced the recoverable population by almost 2 logs from the starting population; this can be attributed to physical removal of organisms from the apple surface. No sanitizer, when used at the recommended concentration, reduced the recovered E. coli population by 5 logs under the test conditions. The most effective sanitizer, peroxyacetic acid, achieved a 5-log reduction when used at 2.1 to 14 times its recommended concentration, depending on the length of the wash time. The chlorine-phosphate buffer solution reduced the population by 5 logs when used at 3 to 15 times its recommended concentration, depending on wash time. At no concentration or wash time tested did chlorine dioxide achieve the 5-log reduction.

scholarly journals Molecular Control of Sucrose Utilization in Escherichia coli W, an Efficient Sucrose-Utilizing Strain

2012 ◽  
Vol 79 (2) ◽  
pp. 478-487 ◽  
Author(s):  
Suriana Sabri ◽  
Lars K. Nielsen ◽  
Claudia E. Vickers
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Good Growth ◽  
Knockout Mutant ◽  
Sucrose Transport ◽  
Molecular Control ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
Sucrose Utilization

ABSTRACTSucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization inEscherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes inE. coliW were examined by knockout and overexpression experiments. At low sucrose concentrations, thecscgenes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout ofcscRandcscKconferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism inE. coliW, demonstrating that no other genes can provide sucrose transport or inversion activities. However,cscKis not essential for sucrose utilization. Fructose is excreted into the medium by thecscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression ofcscA,cscAK, orcscABcould complement the WΔcscRKABknockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressingcscAB, and full growth rate complementation in WΔcscRKABalso requiredcscAB. Our understanding of sucrose utilization can be used to improveE. coliW and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

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.

scholarly journals Rapid Growth of UropathogenicEscherichia coliduring Human Urinary Tract Infection

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Valerie S. Forsyth ◽  
Chelsie E. Armbruster ◽  
Sara N. Smith ◽  
Ali Pirani ◽  
A. Cody Springman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Urinary Tract ◽  
Growth Rates ◽  
High Growth ◽  
Replication Forks ◽  
Chromosomal Replication ◽  
E Coli ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenicE. coli(ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than otherE. coliisolates and survive in that niche. To date, there has not been a reliable method available to measure their growth ratein vivo. Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robustin vivo, matching or exceedingin vitrogrowth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth ratesin vivoat 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR,E. coliin urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth ratesin vivoand resistance to the innate immune response appear to be critical phenotypes of UPEC strains.IMPORTANCEUropathogenicEscherichia coli(UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized byE. colito colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measurein vivogrowth rates of other bacterial pathogens during host colonization.

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