Role of the electrochemical proton gradient in genetic transformation of Haemophilus influenzae.

The uptake of homologous DNA by Haemophilus influenzae was studied as a function of the proton motive force in completely competent cultures in the pH range of 6 to 8. The composition and magnitude of the proton motive force were varied by using the ionophores valinomycin and nigericin (in the presence of various potassium ion concentrations) and by using protonophores. No interaction of the ionophores with the DNA transformation system itself was observed. Either component of the proton motive force, the electrical potential or the pH gradient, can drive the uptake of DNA, and the extent of the uptake of DNA is ultimately determined by the total proton motive force. The transformation frequency increases with the proton motive force, which reaches a maximum value at around -130 mV. These results are consistent with an electrogenic proton-DNA symport mechanism, but direct evidence for such a system is not available. The proton motive force was followed during competence development of H. influenzae at pH 8. In the initial phase (up to 50 min), the proton motive force remained constant at about -90 mV, whereas the transformation frequency rose steeply. In the second phase, the proton motive force increased. The transformation frequency in this phase increased with the proton motive force, as in completely competent cultures. These observations and the observed inhibition by NAD of both the proton motive force and the transformation frequency indicate that structural components of the competent state are formed in the initial phase of competence development, whereas the second phase is characterized by an increase of the proton motive force.     

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.     

不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

Control of transformation competence in Azotobacter vinelandii by nitrogen catabolite derepression.

Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.     

Periodate inhibition of transformation and competence development in Haemophilus influenzae

Ranhand, Jon M. (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Periodate inhibition of transformation and competence development in Haemophilus influenzae. J. Bacteriol. 92:956-959. 1966.-Periodate treatment of competent cells reduced the frequency of transformation to streptomycin resistance about 90% while reducing cell viability about 30% or less. Moreover, when periodate was added to cells early in the competence-development phase, these, too, were unable to develop maximal competence. Periodate inhibition was dependent on time and concentration as well as on the composition of the suspending menstruum. Periodate had no effect on transforming deoxyribonucleic acid (DNA), nor did it prevent transformation when added to competent cells which had already reacted with DNA. Furthermore, the progeny from cells inactivated 90% could be made fully competent, showing that the inhibition was not genetic. It was concluded that the periodate-sensitive substrate may involve the DNA binding site(s).     

Intercellular Effects on Development of Competence in Bacillus subtilis

The intercellular transfer of competence during growth under the conditions specified by the transformation procedure of Spizizen was investigated with Bacillus subtilis 168. The rate of competence development as assayed uniformly in medium B was not affected by variations in the cell concentration, although the first appearance of transformants occurred earlier with high cell densities in medium A, approximately in proportion to the onset of the stationary phase in the culture. Growth in the presence of Pronase enhanced the frequency of transformation, but did not detectably alter the kinetics of competence development. The rate of competence increase in physiologically noncompetent cultures was not changed by mixing with competent cultures either in medium A or in medium B; however, an early appearance of transformants was noted in mixed cultures in which the proportion of competent to noncompetent cells prevented exponential growth of the noncompetent strain. These experiments indicate that the normal development of competence in B. subtilis is not mediated by a soluble or loosely bound protein factor capable of transmitting competence directly via cell contact. The onset of competence is thus a function of internal physiological changes which are induced by the overall metabolic state of the culture.     

Effect of filtrates from transformable and nontransformable streptococci on the transformation of streptococci

Perry, Dennis (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Effects of filtrates from transformable and nontransformable streptococci on the transformation of streptococci. J. Bacteriol. 91:2216-2222. 1966.-The nature of the transformation competence factor from a group H streptococcus was investigated. The activity of competence factor reached a maximum at the time that optimal competence was attained, the maxima of both occurring in the early log phase of growth. The decrease in competence factor was much more gradual than the decrease in number of competent cells. No inhibitor, however, was detected as being responsible for the decrease in either competent cells or competence factor activity. Efforts to induce transformation in other serological groups of streptococci with the use of group H competence factor were unsuccessful. The development of competence in group H when grown in the presence of nontransformable group A strains resulted in a significant increase in the number of transformants. Culture filtrates from early log phase group A cells also caused an increase in the number of transformants from the group H strain. The addition of 10(-4)m ethylenediaminetetraacetic acid to group A (or group H) culture filtrates caused significant increases in the number of transformants. These results thus indicate that group A streptococci, although nontransformable, produce low levels of "competence factor." Late culture filtrates from the group H streptococcus and several strains of group A streptococci possessed deoxyribonuclease-like activity which inhibited the transformation of the group H strain. This activity in the A filtrates, however, was not prevented by group A anti-deoxyribonuclease sera. Instead, these sera also inhibited transformation. Evidence indicates that the lack of transformation of group A streptococci is due to factors other than the production of deoxyribonuclease.     

Deoxyribonucleate Binding and Transformation in Rhizobium japonicum

Rhizobium japonicum, capable of binding high-molecular-weight donor (32)P-labeled deoxyribonucleic acid (DNA) during late log phase in a competence medium, was transformed for streptomycin resistance with a frequency of transformation ranging between 0.02 and 0.08%. Eight to 10% of the homologous native (32)P-labeled input DNA was bound irreversibly in a temperature-dependent manner. Homologous denatured (32)P-labeled DNA was incapable of binding to the recipient under similar conditions. CsCl density gradient banding of the donor and recipient DNA indicated homology. The low frequency of transformation could be due to one or more steps that occur between DNA uptake and integration.     

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.     

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.     

感受态还是芽胞？细胞命运决定的遗传调控网络

枯草芽胞杆菌作为一般认为安全(GRAS,Generally recognized as safe)菌株,被广泛应用于饲料、食品、生物防治等领域,同时,枯草芽胞杆菌作为表达宿主在工业酶的应用中扮演重要角色。然而,低效的芽胞形成率与感受态效率极大限制了枯草芽胞杆菌的应用潜力。尽管已有大量关于芽胞形成与感受态形成的分子遗传机制的研究报道,但是通过遗传改造提高枯草芽胞杆菌芽胞形成率与感受态效率的研究报道并不多。可能的原因是芽胞形成与感受态形成作为枯草芽胞杆菌生长后期两个主要的发育事件,受胞内复杂的遗传调控机制操纵,且两个遗传通路之间存在相互调控关系,对遗传改造工作形成挑战。随着基因工程与代谢工程研究的不断发展,积累了大量关于细胞生长、代谢与发育等方面的遗传信息,通过综合这些遗传信息构建细胞遗传调控网络,用于指导生产实践,已经成为当前研究的热点之一。基于此,本文简要概述了枯草芽胞杆菌芽胞形成和感受态形成的遗传通路,初步探讨了芽胞形成与感受态形成之间的遗传调控网络,及细胞在生长后期的遗传决定机制,并讨论了该遗传调控网络对枯草芽孢杆菌及其近缘种应用研究的指导作用。     

Development of Competence in the Bacillus subtilis Transformation System

Competence in Bacillus subtilis, assayed by the ability of cells to be transformed with bacterial deoxyribonucleic acid (DNA) or transfected by phage DNA, has been shown to occur in a single semisynthetic medium with peak activity occurring 3 hr after the cessation of logarithmic growth. No step-down conditions or culture manipulations were necessary for routine transfection of 1% of the population. The results demonstrate that bacteriophage DNA is a valid assay for studying the development of competence in B. subtilis. Predictions of workers using transforming bacterial DNA, who have suggested that competence in B. subtilis is associated with a specific phase of growth, are substantiated. The peak of competence is not affected by marked differences in the rate of growth during the logarithmic phase. The effect on development of competence by this procedure of some components (including casein hydrolysate, tryptophan, and histidine) which were routinely included in the transformation medium by other investigators has been determined by use of infectious phage DNA as an assay. We have demonstrated that tryptophan, as well as histidine, increases the transformation frequency—even in strains which do not have auxotrophic demands for these components. Glutamic acid and alanine depress optimal levels of transfection.     

Influence of the spxB gene on competence in Streptococcus pneumoniae

In Streptococcus pneumoniae expression of pyruvate oxidase (SpxB) peaks during the early growth phase, coincident with the time of natural competence. This study investigated whether SpxB influences parameters of competence, such as spontaneous transformation frequency, expression of competence genes, and DNA release. Knockout of the spxB gene in strain D39 abolished spontaneous transformation (compared to a frequency of 6.3 × 10⁻⁶ in the parent strain [P < 0.01]). It also reduced expression levels of comC and recA as well as DNA release from bacterial cells significantly during the early growth phase, coincident with the time of spontaneous competence in the parent strain. In the spxB mutant, supplementation with competence-stimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 × 10⁻²). This speaks against the role of SpxB as a necessary source of energy for competence. Neither supplementation with CSP-1 nor supplementation with the SpxB products H₂O₂ and acetate altered DNA release. Supplementation of the parent strain with catalase did not reduce DNA release significantly. In conclusion, the pneumococcal spxB gene influences competence; however, the mechanism remains elusive.     

Induced Natural Transformation of Acinetobacter calcoaceticus in Soil Microcosms

Factors affecting natural transformation of Acinetobacter calcoaceticus BD413 with homologous chromosomal DNA in a silt loam soil microcosm were investigated. Inducible transformation of declining populations of noncompetent A. calcoaceticus cells was detectable for up to 6 days when a simple carbon source, salts, and freshly added DNA were used. In two different experimental setups, the residence time in soil of induced cells could be increased to either 11 or 24 h before DNA addition without reduced transformation frequency; 200-to 1,000-fold fewer transformants were observed following the addition of water. These observations suggest that A. calcoaceticus remains transformable for several hours after its activation by nutrients in soil. Increasing the levels of phosphate salts significantly enhanced the numbers of transformants without increasing the recipient counts correspondingly. Variable levels of ammonium or divalent cations (Mg(sup2+) and Ca(sup2+)) did not have a similar major influence. Soil moisture content significantly affected the transformation frequency of A. calcoaceticus cells, with a general tendency of higher frequencies in drier soil. A minimal frequency was observed at around 35% soil moisture. The data indicate that A. calcoaceticus cells in soil which cannot be detectably transformed are easily induced by nutrients to undergo natural transformation with chromosomal DNA. Access to nutrients seems to be critical for the development and maintenance of competence in soil, which is also affected by abiotic factors like moisture level and phosphate salt concentration.     

Multiple phases of competence occur during the Helicobacter pylori growth cycle

The gastric pathogen Helicobacter pylori undergoes genetic exchange at unusually high frequencies, primarily through natural transformation. Despite progress toward understanding the molecular mechanism of natural transformation in H. pylori, little is known about how competence is regulated or its relationship to DNA release. By measuring transformation incrementally throughout the growth curve, we show that H. pylori exhibits a novel pattern of competence with distinct peaks of transformation during both logarithmic and stationary growth phases. Furthermore, different H. pylori strains vary in the presence and timing of their competence peaks. We also examined the process of DNA release in relation to competence. Although extensive DNA release does not occur until late stationary phase, sufficient genomic DNA was present during the logarithmic phase to yield measurable transformants. These results demonstrate that the state of competence in H. pylori occurs in an unprecedented pattern during the growth curve with no clear relationship to DNA release.     

Sensitivity of a human hybrid cell line (HeLa X skin fibroblast) to radiation-induced neoplastic transformation in G2, M, and mid-G1 phases of the cell cycle

The dependence of gamma-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa X skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 or for asynchronous cells. However, the data indicate that the transformation frequencies were similar in the different phases of the cell cycle when correlated with surviving fraction. The results indicate that transformation frequency is more sensitive to changes in dose than is cell survival.     

Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.