Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

Purification of Competent Cells in the Bacillus subtilis Transformation System

Transformed cells have been separated from nontransformed cells by centrifugation on a density gradient of Renografin-76. Separation was achieved both on a linear gradient and on a discontinuous gradient. Under optimal conditions, all of the cells in one band (median density, 1.110 g/ml) were transformants, whereas virtually all cells in the other (median density, 1.131) were nontransformants. In some instances, recentrifugation of the transformant band further enriched the transformant population. The transformed population can also be enriched by zonal centrifugation in a linear gradient of Ficoll. However, this technique is far less efficient than centrifugation in Renografin-76. Since the density of competent cells is identical to that of transformants, we conclude that the low density is a property of competent cells. The significance of this low density to the physiology of competent cells is discussed.     

Uptake of Synthetic Polynucleotides by Competent Cells of Bacillus subtilis

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.     

Structure and Replication of Chromosomes in Competent Cells of Bacillus subtilis

Competent cultures of Bacillus subtilis 168 were fractionated on gradients of Renografin-76 to obtain a population enriched for competent cells. The cells in this fraction contained two nuclear bodies. The competent cell fraction synthesized deoxyribonucleic acid and ribonucleic acid at reduced rates compared to the noncompetent cell fraction and appeared to divide synchronously upon incubation. The state of the chromosome in competent cells was determined by density transfer experiments and marker frequency analyses. The results are consistent with a competent cell possessing two, or a multiple of two, chromosomes, one complete and the other partially duplicated. During subsequent growth the partially completed chromosome replicates preferentially.     

Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.     

Deoxyribonucleic Acid Repair in Bacillus subtilis: Development of Competent Cells into a Tester for Carcinogens

The development of competent transformed Bacillus subtilis into a tester system for carcinogens is described. Precocious or noninduced activation of SOS functions occur in competent cells. Thus, lower doses or concentrations of SOS inducing agents are needed to cause cell death due to indigenous prophage activation and lysis of bacteria. The two known defective prophages in B. subtilis enhance the sensitivity of competent cells to the carcinogens ultraviolet light, mitomycin C, and methyl methanesulfonate. However, these same cells have no enhanced sensitivity for the non-carcinogenic ethyl methanesulfonate or for nalidixic acid. Therefore, competent B. subtilis appear to be a sensitive tester for carcinogens.     

Increase in Lytic Activity in Competent Cells of Bacillus subtilis After Uptake of Deoxyribonucleic Acid

Extractable lytic activity in competent cells of Bacillus subtilis 168 was markedly increased after treatment with homologous or heterologous deoxyribonucleic acid (DNA). This increase was prevented by deoxyribonuclease, and did not occur with B. subtilis W23 or with noncompetent B. subtilis 168 cells, neither of which take up DNA. Although the deoxyribonuclease-sensitive step in DNA uptake was completed within 10 min, the increase in lytic activity did not begin until more than 30 min after the addition of DNA. The increase was prevented by any of several antibiotics. These results are discussed in relation to the mechanisms for the uptake of transforming DNA and the lysis of transfected cells.     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

水平基因转移

简要介绍了水平基因转移（horizontal gene transfer,HGT）的基本概念以及进行转移的主要方式：由质粒或病毒等介导的水平基因转移和基因的“直接”水平转移。并结合基因组序列分析,重点阐述远缘生物之间发生的广泛的基因交流,以及其与进化、系统发育和基因工程生物安全性问题的探讨。 Abstract:In this paper the conception of horizontal gene transfer (HGT) was introduced,and main mode of HGT was also enumerated as follows:HGT by medium such as plasmid and virus etc.and the HGT without any medium.The transfer of genes from one species to another especially between remote species was emphasized by the information from genome sequencing.The problems about evolution phylogenies and safety of GEMs (gene engineered microorganisms) for HGT were discussed.     

Genetic Analysis of Repair of Ultraviolet Damage by Competent and Noncompetent Cells of Bacillus subtilis

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T(-) has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.     

Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

The Tryptophanase Gene Cluster of Haemophilus influenzae Type b: Evidence for Horizontal Gene Transfer

Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS.     

植物中的水平基因转移

水平基因转移是不通过生殖而进行的遗传物质交流, 在原核生物和单细胞真核生物的进化中起着重要作用。然而, 水平基因转移在多细胞真核生物之间的发生频率以及对多细胞真核生物进化的影响尚不明确。近期的一些研究显示, 水平基因转移在高等植物之间以及高等植物和其它生物之间普遍存在。该文将对高等植物中已发现的一些水平基因转移现象进行综述, 并尝试解析植物之间水平基因转移可能的机制及其重要意义。     

Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis

Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacterium Bacillus subtilis by constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR-D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator Spo0A～P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation of yuxH by Spo0A～P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional c-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.     

Macromolecular Synthesis in Bacillus subtilis During Development of the Competent State

Rates of deoxyribonucleic acid, ribonucleic acid, and protein synthesis were examined in purified competent cells of Bacillus subtilis during the development of the transformable state. To become competent, a cell must depart from the normal course of vegetative growth and pass through a precompetent phase beginning as early as 90 to 180 min before the appearance of transformability. While in the precompetent state, the cell decreases its rate of deoxyribonucleic acid synthesis and lowers its ratio of ribonucleic acid synthesis to protein synthesis. This altered pattern of synthesis eventually leads to a decreased buoyant density of precompetent cells. Once a cell has become both precompetent and low in density, it can be converted to a competent (transformable) cell. The early alterations in macromolecular synthesis were found in two competence regimens, one utilizing a nutritional step-down and one free of such a shift. The data imply that the precompetent state is a generalized characteristic of the B. subtilis transformation system and is not specific to the procedure used to allow competence development. Since precompetence-specific events occur very early in a competence regimen, we conclude that the induction of precompetence is unrelated to sporulation or a nutritional shift.     

Binding of Rabbit Gamma Globulin by Competent Bacillus subtilis Cultures

Deoxyribonucleic acid (DNA)-mediated transformation of Bacillus subtilis can be inhibited by antibodies which specifically interact with single-stranded DNA. This inhibition occurs at a time when the transformation reaction is insensitive to deoxyribonuclease. Studies with radioactive proteins revealed that the maximal binding of gamma globulin occurs immediately preceding the development of maximal competence in the population. Other proteins, such as deoxyribonuclease cytochrome c and serum albumin also adsorb to the surface of the cell. After treatment with lysozyme, 67% of the radioactive gamma globulin remains associated with the cytoplasmic membrane. These findings suggest that the DNA is complexed in a deoxyribonuclease-insensitive form to the surface of the cell and is converted to a single-stranded state prior to transport past the membrane and integration into the chromosome.     

All Seven comG Open Reading Frames Are Required for DNA Binding during Transformation of Competent Bacillus subtilis

The seven proteins encoded by the comG operon of Bacillus subtilis exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in comG result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed.