Coli K12 Cells Research Articles

Phagocytosis and oxidative activity of neutrophils after interaction with uropathogenic <i>Escherichia coli</i> biofilms

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux+ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p < 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

Bacterial electroactivity and viability depends on the carbon nanotube-coated sponge anode used in a microbial fuel cell

The anode material is vital to improve the power generation of a microbial fuel cell (MFC). In this study, a carbon nanotube (CNT)-coated sponge with macro-porous structure, high surface area, and high conductivity was constructed as an anode to encapsulate Escherichia coli K12 (E. coli K12) cells. To achieve high power generation of the MFC, the optimal concentration of the CNT coating the sponge was found to be 30mgmL−1. At this concentration, a maximum power density of 787Wm−3 and a chemical oxygen demand (COD) removal of 80.9% were obtained with a long stable electricity generation process in batch mode. This indicates that the biofilm on the CNT (30mgmL−1)-coated sponge possessed excellent electroactivity and stability. Scanning electron microscope (SEM) images confirmed that the CNT-coated sponge provided a suitable microenvironment for E. coli K12 cells to maintain their attachment and colonization. Additionally, a CNT-dependent viability phenomenon of the E. coli K12 cells was discovered after electricity generation. This CNT-dependent viability of the E. coli K12 cells was stable and sustainable after storage at −20°C in a milk tube for one year.

Microcin H47 System: An Escherichia coli Small Genomic Island with Novel Features

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island –H47 genomic island- that would employ a parasitic strategy for its mobility.

Dynamic viscoelastic behavior of individual Gram-negative bacterial cells

We have used atomic force microscopy (AFM) to measure the viscoelastic properties of individual Escherichia coli K12 cells under fully hydrated conditions by collecting AFM force–indentation and force–time curves. Spherical colloidal tips were used to reduce the local strain ensuring that the measurements were performed in the linear viscoelastic regime. We find that the cells exhibit a time-dependent viscoelastic response to the constant compressive force applied by the AFM tip. The results are interpreted using the standard solid model which describes both an instantaneous and a delayed elastic deformation. The values obtained for the three viscoelastic parameters were used to generate a full description of the dynamic viscoelastic behavior of the cells over a wide range of frequencies derived from the differential equation governing the standard solid model. The results obtained using this methodology are compared to experimental data obtained for the E. coli K12 cells and these results are compared to previously reported viscoelastic data on Pseudomonas aeruginosa PAO1 cells. The differences between the viscoelastic behaviors of these two types of Gram-negative bacterial cells are discussed in terms of their structural differences.

L-methionine γ-lyase from Citrobacter freundii: Cloning of the gene and kinetic parameters of the enzyme

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.

Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new Flavin:NAD(P)H reductase subfamily.

Escherichia coli W uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by the hpaB and hpaC genes, respectively, that form a single transcription unit. Overproduction of the small HpaC component in E. coli K-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochrome c and ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin of Streptococcus pneumoniae allowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase of E. coli W as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.

Bactericidal activity of photocatalytic TiO(2) reaction: toward an understanding of its killing mechanism.

When titanium dioxide (TiO(2)) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO(2) photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol. mg (dry weight) of cells(-1) after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO(2). We concluded that TiO(2) photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.

Electrochemical measurements of glucose dehydrogenase activity exhibited by Escherichia coli cells; effects of the additions of pyrroloquinoline quinone, magnesium or calcium ions and ethylenediaminetetraacetic acid

An electrode modified with immobilized Escherichia coli K12 cells produces a steady-state catalytic current originating from the glucose dehydrogenase activity (GDH) of the bacterial cells. Thus, the catalytic current provides a convenient means of measuring the bacterial GDH activity. E. coli K12 cells cultivated under ordinary conditions exhibit a GDH activity, the magnitude of which is about 15% of the full activity attained in the presence of pyrroloquinoline quinone (PQQ) and magnesium ions. The E. coli K12 cells mostly lose the full GDH activity when treated with ethylenediaminetetraacetic acid, and restore the activity by the additions of PQQ and magnesium or calcium ions. The rate and degree of the restoration of GDH activity strongly depend on the concentrations of PQQ and the divalent cations. The concentration dependence of the bacterial GDH activity is similar to that of the catalytic activity of purified apo-GDH except that the bacterial GDH activity is fully restored by calcium ion as well as magnesium ion, while catalytic activity of apo-GDH is only partially restored by calcium ion.

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by (i) exposure level, (ii) organ distribution of (DNA) dose, and (iii) genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenicity potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria ( E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG > ENU ⩾ DES > DEN ≅ EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations. The results also indicated that O 6-ethylguanine was the DNA adduct most probably responsible for mutagenesis both in the bacteria and the mammalian cells. Comparative mutagenesis experiments performed with the E. coli cells in vitro with and without mammalian liver preparations (mouse S9) and in animal-mediated assays, in which the E. coli indicators were present in livers of treated mice, gave the following results: The addition of mouse liver S9 allows the detection of activating (DEN) or deactivating (ENNG) effects of mammalian metabolism, but the ranking order of mutagenic potency remained the same, namely ENNG > ENU > DES > EMS ≅ DEN. In the animal-mediated assays, on the other hand, the order of mutagenic potency was quite different, namely, DEN > ENU > ENNG > EMS. This latter order of activity is similar to the order of carcinogenicity of these compounds in rodents, and probably also reflects the degree of DNA alkylation in liver cells of the treated animals. These and other earlier results indicate that the relative ranking order of mutagenic potency observed in vitro, regardless whether it was obtained with bacterial or mammalian cells, is not representative of the situation in vivo. Although the addition in in vitro assays of mammalian liver preparations allows the detection of those compounds which are likely to be activated or deactivated in vivo, it appears that the magnitude of the effect is not adequately represented in the in vitro assays. In search for rapid methods for estimating genotoxic factors in various organs in vivo, use was made of the DNA repair host-mediated assay. In this test, repairable DNA damage is measured in pairs of E. coli K12 cells which differ by their ability of repairing lethal DNA damage, and which can be recovered from stomach, intestine, colon, liver, spleen, lungs, kidneys, testes and the blood of treated rodents (mice). Preliminary experiments using a variety of differently acting compounds have indicated that this assay procedure may become useful in rapidly assessing the presence and persistence of genotoxic factors in various organs of mice; its general usefulness as genotoxic dose monitor in vivo is presently being investigated.

Potentiation of Antibiotic Bactericidal Activity by Normal Human Serum

Combinations of certain antibiotics and normal human serum at concentrations at which there was no killing by the agents when used alone were found to be bactericidal for Escherichia coli K-12 cells. This effect was observed with tetracycline, streptomycin (SM), trimethoprim, and ampicillin, but not with chloramphenicol or nalidixic acid. Synergy between SM and human serum was also observed against four of nine smooth strains of E. coli. A plasmid-bearing strain of E. coli K-12 was also killed by combinations of tetracycline or SM plus serum, even though the plasmid conferred resistance to tetracycline and SM. Evidence is presented that the synergy between antibiotics and serum is due to a complement-mediated effect on the bacterial cells that makes the cells more susceptible to the bactericidal effects of the antibiotics.

Glutamate transport in Escherichia coli K-12: nonidentity of carriers mediating entry and exit.

The exit of glutamate from Escherichia coli K-12 cells preloaded with the radioactive amino acid and its relation to the reaction of entry were studied. Experiments with cells preloaded to different intracellular concentrations of radioactive glutamate confirmed our earlier conclusion that glutamate exit was a first-order reaction. l-Glutamate, competitive inhibitors of glutamate uptake (d-glutamate and l-glutamate-gamma-methyl ester), noncompetitive inhibitors of glutamate uptake (l-serine and l-alanine), and the energy poison NaN(3) all accelerated glutamate exit 2.8-fold. No additive effect was observed in the presence of NaN(3) together with l-glutamate. Preloading with cold l-glutamate did not increase the rate of uptake of radioactive glutamate. It is concluded that the acceleration of glutamate exit in the presence of l-glutamate in the medium is not due to exchange diffusion and that l-glutamate and azide affect exit indirectly by preventing recapture. Sucrose, 25%, slowed down glutamate exit by a factor of about 4.7 and increased the steady-state level of glutamate accumulation to about the same extent. Increasing the intracellular K(+) concentration enhanced glutamate uptake but did not affect the half-time of exit. It is concluded that separate carriers are most probably involved in mediating the entry and exit reactions.

Isolation and characterization of an Escherichia coli bacteriophage requiring cell wall galactose.

A new coliphage, designated U3, has been selected for the ability to discriminate the presence of galactose in the cell wall of Escherichia coli. U3 attacks E. coli K-12 cells that are able to incorporate galactose into their cell walls, but mutants blocked in the synthesis of uridine diphosphogalactose, the precursor of cell wall galactose, are completely resistant to the phage. U3 is a small, tail-less, approximately spherical phage resembling phiX174 in its physical properties. Its diameter by electron microscopy is 21 to 22 nm, and its particle weight is approximately 4 x 10(6) daltons. Like phiX174, U3 appears to have a single-stranded deoxyribonucleic acid genome and has at least four cistrons.