Exploring permeability of Escherichia coli competence using quantum dots as fluorescent probes

Though people had recognized the pivotal function of CaCl(2) during DNA transformation into Escherichia coli, the mechanism of divalent Ca(2+) cation inducing E. coli competence development is still unknowable. Quantum dots (QDs), as a new fluorescent probe, being applied in biology research, had aroused great interest. We explored the penetrability of E. coli competent cells membrane using QDs and proved directly that competent cells were more permeable than that of noncompetent. The results are significant on understanding the problems of the microbiological genetics.     

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.     

Calcium regulation of growth and differentiation in Streptococcus pneumoniae.

Streptococcus pneumoniae requires 0.15 mM-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mM triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2',4'-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。     

Transformation of colonial Escherichia coli on solid media

We report that colonial Escherichia coli cells on various solid media can develop modest genetic competence. Using an on-filter culture system, we found that E. coli colonies on CaCl2-containing agar were transformed in the presence of plasmid DNA. Interestingly, transformation also occurred on LB-agar, various moist foods and even on H2O-agar. These results suggest that some populations of colonial E. coli in various environments could become transformable regardless of the surrounding Ca2+ concentration.     

Escherichia coli cell competence induced by calcium cations]

Escherichia coli K-12 cells grown to early and late exponential, and early and late stationary phases were treated with CA2+ and analysed for the ability of exogenous 14C-DNA uptake and genetic transformation. DNA-membrane complexes were detected detected by isopicnic centrifugation of cell lysates in sucrose density gradient. It is found that 1) during all the tested phases of the growth cycle, E. coli cells attain the ability of enhanced DNA uptake after Ca2+ treatment; 2) exogenous and host DNAs are associated with the cell membrane during all tested growth phases; 3) nevertheless, the ability to form transformants is strongly time-dependent: the cells can be transformed during logarithmic phase only; 4) Ca2+ protects exogenous DNA against its degradation by bovine pancreatic DNAase. The peculiarities of Ca2+-induced competence, actual and possible interference of Ca2+ at different transformation steps are briefly discussed.     

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2-mediated transformation process

In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl2 (50-100 mM). It is believed that CaCl2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E. coli cell surface; however, the binding mechanism is mostly obscure. In this report, we present our findings of an in-depth study on in vitro interaction between plasmid DNA and E. coli LPS, using different techniques like absorption and circular dichroism spectroscopy, isothermal titration calorimetry, electron and atomic force microscopy, and so on. The results suggest that the Ca(II) ions, forming coordination complexes with the phosphates of DNA and LPS, facilitate the binding between them. The binding interaction appears to be cooperative, reversible, exothermic, and enthalpy-driven in nature. Binding of LPS causes a partial transition of DNA from B- to A-form. Finer study with the hydrolyzed products of LPS shows that only the core oligosaccharide domain of LPS is responsible for the interaction with DNA. Moreover, the biological significance of this interaction becomes evident from the observation that E. coli cells, from which the LPS have been leached out considerably, show higher efficiency of transformation, when transformed with plasmid-LPS complex rather than plasmid DNA alone.     

Genetic transformation in freshwater: Escherichia coli is able to develop natural competence.

Until now, Escherichia coli was thought to be unable to develop natural competence, i.e., genetic transformation could be achieved only artificially with the aid of nonphysiological concentrations of calcium ions or by other treatments. We have tested the competence development of E. coli through transformation under natural conditions in river water, springwater, and mineral water which contained between 0 and 11 mM Ca2+, using pUC18 DNA. The presence of calcium ions at concentrations as low as 1 to 2 mM was sufficient to obtain transformants. Variations in the temperature of incubation were not required for competence development but had an influence on the transformation frequency. Using water from mineral springs originating from calcareous regions, we have obtained transformation frequencies with laboratory strains of E. coli similar to those reported for other gram-negative bacteria known to develop natural competence. The competence development of E. coli is most probably internally regulated (as for the other gram-negative bacteria), and inadequate conditions chosen for the transformation tests in the laboratory might impair the detection of higher natural transformation frequencies. The results will enhance our knowledge about the fate of laboratory or production strains of E. coli cells reaching natural aquatic ecosystems.     

The ability to develop an activity that transfers histones onto sperm chromatin is acquired with meiotic competence during oocyte growth.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, transferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to SrCl2. One-third of treated oocytes produced at least one Ca2+ oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the Ca2+ chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a Ca2+-dependent process.     

Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types

All pancreatic endocrine cells, producing glucagon, insulin, somatostatin, or PP, differentiate from Pdx1+ progenitors that transiently express Neurogenin3. To understand whether the competence of pancreatic progenitors changes over time, we generated transgenic mice expressing a tamoxifen-inducible Ngn3 fusion protein under the control of the pdx1 promoter and backcrossed the transgene into the ngn3(-/-) background, devoid of endogenous endocrine cells. Early activation of Ngn3-ER(TM) almost exclusively induced glucagon+ cells, while depleting the pool of pancreas progenitors. As from E11.5, Pdx1+ progenitors became competent to differentiate into insulin+ and PP+ cells. Somatostatin+ cells were generated from E14.5, while the competence to make glucagon+ cells was dramatically decreased. Hence, pancreas progenitors, similar to retinal or cortical progenitors, go through competence states that each allow the generation of a subset of cell types. We further show that the progenitors acquire competence to generate late-born cells in a mechanism that is intrinsic to the epithelium.     

Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.     

Imaging Escherichia coli using functionalized core/shell CdSe/CdS quantum dots

The internalization of a series of water-soluble CdSe/CdS quantum dots (QDs) stabilized by citrate, isocitrate, succinate, and malate by Escherichia coli is established by epifluorescence and confocal fluorescence scanning microscopy, fluorimetry, and UV–vis spectroscopy on whole and lysed bacterial cells. The organic-acid-stabilized QDs span a range in size from 3.8±1.1 to 6.0±2.4 nm with emission wavelengths from 540 to 630 nm. QDs of different sizes (i.e., 3.8–6 nm) can enter the bacterium and be detected on different fluorescence channels with little interference from other QDs as a result of the distinct emission profiles (i.e., 540–630 nm, respectively). Costaining QD-labeled E. coli with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) demonstrates that the QDs and DAPI are colocalized within E. coli, whereas costaining QD-labeled E. coli with membrane dye FM4-64 shows that the FM4-64 is localized in the outer bacterial membrane and that the QDs are inside.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Infection of Actinomycin-Permeable Mutants of Escherichia coli with Urea-Disrupted Bacteriophage

Intact cells of actinomycin-permeable mutants of Escherichia coli could be infected with urea-disrupted phage T4 (designated as T4pi). The parental strains and the revertants, which are impermeable to actinomycin, were not susceptible to T4pi unless they had been treated with agents which altered their permeability. The permeable mutants developed competence for pi infection during the growth cycle; cells in the early stationary phase produced 10- to 100-fold more plaques on plating with T4pi than did exponentially growing cells. Colistin (polymyxin E) was effective in converting noncompetent cells of either permeable or nonpermeable strains to the competent state. Treatment with lysozyme resulted in a considerable increase in susceptibility to T4pi of permeable mutants but not of nonpermeable cells. It appears that development of competence for pi infection is mainly due to alterations in the permeability barriers of the cell.     

Factors affecting transformation of Bacillus licheniformis

Thorne, Curtis B. (Fort Detrick, Frederick, Md.), and Harold B. Stull. Factors affecting transformation of Bacillus licheniformis. J. Bacteriol. 91:1012-1020. 1966.-Transformation systems involving two types of transformable mutants of Bacillus licheniformis 9945A were compared. Each system required its specific growth medium, but a single transformation medium could be used for both. Cells from a culture of optimal age were not competent, at least to any great extent, but they developed competence during incubation in a transformation medium. With each system, 3 to 5% of the recipient cells were transformed upon exposure to wild-type deoxyribonucleic acid (DNA) for 2 to 3 hr. When competent cells were exposed to DNA for 30 min, 1 to 2% of them were transformed. The data are interpreted to mean that cells were heterogeneous with respect to development of competence, and when properly grown cells were incubated in transformation medium some of them gained competence, whereas others lost it. If DNA was present during the entire period, the cells were transformed as they became competent and the transformants accumulated. However, during any short period of exposure to DNA, only those cells that were competent at the time were potential transformants. The high frequencies of transformation obtained in these studies made it feasible to prepare marked strains by transforming markers into recipient cells. These experiments demonstrated that the characteristics of the two transformation systems could not be attributed to specific nutritional markers. Presumably, each of the two series of highly transformable auxotrophic mutants also carried at least one other mutation that resulted in development of competence under the specific conditions.     

Penetration of exogenous DNA through the membranes of Escherichia coli treated with Ca2+ cations]

Possible role of electrochemical potential as driving force for exogenous DNA penetration inside Ca2+ treated Escherichia coli was investigated using carbonyl-cyanide-m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. CCCP at concentrations of 10(-6) -10(-5) M did not affect the number of plague forming units. The inhibitory effect was observed under higher concentrations (5.10(-5) -10(-4). This effect was not due to the loss of cell viability and is attributed to the reduced capacity of the cells to interact with DNA. It is suggested that conformational changes in biomembranes might be at least partially involved. It is concluded that the electrochemical potential is not the driving force for penetration of exogenous DNA inside Ca2+ -treated E. coli cells. Bronian movement is suggest as an alternative.     

Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii.

Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-).     

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.     

Investigation of the neutral filter elution technique I. Effects of pore density and pore diameter on elution rate at pH 9.6

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.