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1.
Isolation of the Escherichia coli nucleoid 总被引:4,自引:0,他引:4
Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size of either nucleoid. 相似文献
2.
J R Pellón 《Revista Espanola de Fisiología》1983,39(3):321-325
Changes in the structure of the Escherichia coli nucleoid during heat damage and repair were followed by sedimentation analysis in neutral sucrose gradients. Heating at 50 degrees C results first in a decrease in the sedimentation coefficient of the isolated nucleoid. Increasing the heating time, a subsequent increase in sedimentation coefficient is observed. After a heat shock (i.e. 4 min at 50 degrees C), a short incubation at 25 degrees C (i.e. 5 min) allows the nucleoid to repair and return to the sedimentation coefficient of control unheated nucleoids. The nucleoids heated at 50 degrees C for longer periods and incubated afterwards at 25 degrees C demonstrate a different pattern of structural repair. They associate with protein in the first stage of the repair period. 相似文献
3.
4.
J R Pellon 《The Journal of applied bacteriology》1983,54(3):437-439
The repair of the Escherichia coli nucleoid structure after heat shock (50 degrees C, 5 min) was studied. After heat shock the repair process did not include the association of the nucleoid to protein structures as is the case after more severe heat treatments resulting in cell death or inactivation. 相似文献
5.
J.R. Pellon 《Journal of applied microbiology》1983,54(3):437-439
The repair of the Escherichia coli nucleoid structure after heat shock (50°C, 5 min) was studied. After heat shock the repair process did not include the association of the nucleoid to protein structures as is the case after more severe heat treatments resulting in cell death or inactivation. 相似文献
6.
Effect of rifampin on the structure and membrane attachment of the nucleoid of Escherichia coli 总被引:31,自引:17,他引:14
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Deoxyribonucleic acid (DNA) of Escherichia coli was found to be attached to the cell membrane at about 20 points. This was determined by fractionation of X-irradiated cells with the M band (magnesium-Sarkosyl crystals) technique. The number of attachment points was computed from the relationship between the amount of DNA in M bands and the number of double-strand breaks introduced by the X-ray treatment. The number of attachment points was decreased fourfold by treatment of cells with rifampin. This effect was apparently due to the action of the drug on ribonucleic acid (RNA) polymerase since the drug did not affect a mutant whose RNA polymerase is resistant to rifampin. This suggests that there may be two classes of attachment points of DNA on the membrane, some of which are removed by rifampin treatment and some which are not. Rifampin treatment also resulted in the uncondensing of isolated nucleoids and in an axial appearance of the nucleoids in ultrathin sections. The results suggest that RNA polymerase plays a role, direct or indirect, in maintaining the structure of the bacterial nucleoid and in some of its attachment to the membrane. 相似文献
7.
The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria. Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli. By its properties HU shares some traits with histones and HMG proteins. More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor. For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells. To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining. We show that DAPI and HU-GFP colocalize on the E. coli nucleoid. HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell. 相似文献
8.
Nucleoids obtained from E. coli cells by extraction with 1 M NaCl and detergents containing solution were further extracted with 2 M NaCl. From these samples, that contain only tightly bound proteins, fractions of protein core and peripheral nucleoprotein were obtained. It is shown that DNA synthesis proceeds mainly in the core structures. We have found that DNA polymerase I, which is bound with DNA nucleoid loops and with the above mentioned core structures, is not dissociating in 2M NaCl solution. 相似文献
9.
《中国科学:生命科学英文版》2015,(9)
The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as protein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly compacted than that in the exponential phase. In addition to factor for inversion stimulation(Fis), histone-like nucleoid structuring protein(H-NS), heat-unstable nucleoid protein(HU) and integration host factor(IHF) here we have identified, three new candidates of E. coli nucleoid, namely DNA-binding protein from starved cells(Dps), host factor for phage Qβ(Hfq) and suppressor of td- phenotype A(Stp A). Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, Stp A and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli. 相似文献
10.
Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters. 相似文献
11.
The advantages associated with the described immobilized enzyme reactor include: (1) Use of the common rate equations and kinetic parameters. (2) Detection of significant lag periods. (3) Quantitative measure for non-covalently attached enzyme. (4) The means for washing the immobilized enzyme allows for the repeated use of the same matrix-bound enzyme. (5) Constant temperature control. (6) Both unbound native and matrix-bound enzyme may be reacted under identical conditions. (7) No grinding of the glass-bound enzyme or other matrix fragmentation occurs because no abrasive forces are required for stirring. 相似文献
12.
L J Moberg 《Applied and environmental microbiology》1985,50(6):1383-1387
An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method. 相似文献
13.
Skoko D Yoo D Bai H Schnurr B Yan J McLeod SM Marko JF Johnson RC 《Journal of molecular biology》2006,364(4):777-798
Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending. With increasing concentration, Fis first coats DNA to form an ordered array with one Fis dimer bound per 21 bp and then abruptly shifts to forming a higher-order Fis-DNA filament, referred to as a low-mobility complex (LMC). The LMC initially contains two Fis dimers per 21 bp of DNA, but additional Fis dimers assemble into the LMC as the concentration is increased further. These complexes, formed at or above 1 microM Fis, are able to collapse large DNA molecules via stabilization of DNA loops. The opening and closing of loops on single DNA molecules can be followed in real time as abrupt jumps in DNA extension. Formation of loop-stabilizing complexes is sensitive to high ionic strength, even under conditions where DNA bending-compaction is unaltered. Analyses of mutants indicate that Fis-mediated DNA looping does not involve tertiary or quaternary changes in the Fis dimer structure but that a number of surface-exposed residues located both within and outside the helix-turn-helix DNA-binding region are critical. These results suggest that Fis may play a role in vivo as a domain barrier element by organizing DNA loops within the E. coli chromosome. 相似文献
14.
The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates. 相似文献
15.
The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data. 相似文献
16.
Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy 总被引:5,自引:0,他引:5
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Kim J Yoshimura SH Hizume K Ohniwa RL Ishihama A Takeyasu K 《Nucleic acids research》2004,32(6):1982-1992
A small container of several to a few hundred µm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ~80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid. 相似文献
17.
Kang MS Okuyama M Yaoi K Mitsuishi Y Kim YM Mori H Kim D Kimura A 《The FEBS journal》2007,274(23):6074-6084
The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1-->6)-D-Manp, alpha-D-Xylp-(1-->6)-D-Fruf, and alpha-d-Xylp-(1-->3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-d-Xylp-(1-->6)-D-Manp and alpha-d-Xylp-(1-->6)-D-Fruf inhibited intestinal alpha-glucosidases. 相似文献
18.
A simple filter paper assay for the measurement of Escherichia coli 4-thiouridine-tRNA sulfurtransferase activity is described. The assay includes the following procedures: (a) incubation of enzyme with appropriate substrates including unfractionated yeast tRNA and [35S]cysteine, (b) reisolation of tRNA, and (c) binding of tRNA to ion exchange filter papers. The assay can be routinely performed with relatively small sample volumes (0.1 ml) and completed within 14 h. Proof of the validity of the assay is based in part on two experimental observations: (1) tRNA is the predominant 35S-labeled species remaining bound to the filter after extensive washing, and (2) 4-thiouracyl is the predominant thiolated base formed during the assay. 相似文献
19.
Ken-ichi Yamazaki Akihisa Nagata Yasunobu Kano Fumio Imamoto 《Molecular & general genetics : MGG》1984,196(2):217-224
Summary Of the molecular species of proteins associated with the nucleoids of Escherichia coli cells, those with relatively high affinity to bind to DNA were isolated and characterized. Seven classes of nucleoid proteins with molecular weights of 9,000, 17,000 (two molecular species), 22,000, 24,000, 27,000 and 28,000 were isolated at more than 90% purity or were partially purified. On the basis of its amino acid composition and other chemical properties, the 9,000 dalton protein was identified as HLP II (or HU protein or BH2) (Pettijohn 1982: Rouvière-Yaniv and Gros 1975; Varshavsky et al. 1978). The 17 K protein consisted of two molecular species and one of these, 17 K (a) protein, seemed to be identical with HLPI (or protein 1 or BH1) reported previously (Pettijohn 1982; Varshavsky et al. 1977; Varshavsky et al. 1978). The 26 K protein was identical to the 22 K protein (Kishi et al. 1982). The 27 K protein showed immunological cross-reactivity with the antibody for histone H2A and was thus identified as the H protein reported previously (Hübscher et al. 1980). Two basic proteins, 9 K and 17 K(a), showed relatively high binding affinities to DNA, while the 28 K protein showed moderate binding affinity. The biological significance of these nucleoid proteins, which constitute a family of proteins participating in formation of the nucleoid structure, is discussed. 相似文献
20.
L J Moberg 《Applied microbiology》1985,50(6):1383-1387
An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method. 相似文献