Relationship Between Competence for Transformation of Bacillus subtilis with Native and Single-Stranded Deoxyribonucleic Acid

The response of populations of Bacillus subtilis to both native deoxyribonucleic acid (DNA) and denatured DNA was investigated at maximal competence and at various times during the development of compentency. The results indicate that competence for transformation with native and denatured DNA increases and decreases simultaneously. Competition occurs between native and single-stranded DNA during transformation, and the same cells in a population can be doubly transformed by DNA molecules of both configurations.     

Fate of Transforming Deoxyribonucleate in Bacillus subtilis

The majority of donor deoxyribonucleate (DNA) at early stages after uptake was found in a complex with a cell component which changes its buoyant behavior on equilibrium density gradients. Analysis of the recipient cell lysates, after treatment to dissociate the complex, showed about two-thirds of the donor molecules in denatured form and the rest associated with recipient DNA. Incubation of cells after DNA uptake leads to the disappearance of denatured donor DNA and to the increase of donor label associated with recipient DNA. Some characteristics of a component from intact cells or spheroplasts with affinity for denatured Bacillus subtilis DNA are described.     

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.     

Ubiquitous late competence genes in Bacillus species indicate the presence of functional DNA uptake machineries

Natural competence for genetic transformation, i.e. the ability to take up DNA and stably integrate it in the genome, has so far only been observed in the bacterial kingdom (both in Gram-negative and Gram-positive species) and may contribute to survival under adverse growth conditions. Bacillus subtilis , the model organism for the Bacillus genus, possesses a well-characterized competence machinery. Phylogenetic analysis of several genome sequences of different Bacillus species reveals the presence of many, but not all genes potentially involved in competence and its regulation. The recent demonstration of functional DNA uptake by B. cereus supports the significance of our genome analyses and shows that the ability for functional DNA uptake might be widespread among Bacilli .     

Competence proteins in Bacillus subtilis com mutants

The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.     

Genetic Recombination of Transforming Deoxyribonucleic Acid Molecules with the Recipient Genome and Among Themselves in Protoplasts of Bacillus subtilis

Hirokawa, Hideo (Southwest Center for Advanced Studies, Dallas, Tex.), and Yonosuke Ikeda. Genetic recombination of transforming deoxyribonucleic acid molecules with the recipient genome and among themselves in protoplasts of Bacillus subtilis. J. Bacteriol. 92:455-463. 1966.-Re-extraction of transforming deoxyribonucleic acid (DNA) from protoplasts of Bacillus subtilis is much more efficient than from intact competent cells. This facilitated the detection of physical recombination between donor and recipient DNA molecules, as indicated by a high cotransfer index of ind(+) and his(+) markers which were originally located in exogenous and endogenous DNA molecules, respectively. This recombinant DNA was extracted after 30 min of incubation of ind his(+) protoplasts with ind(+)his DNA, previously extracted from a corresponding mutant strain of B. subtilis. The intracellular formation of recombinant molecules (ind(+)his(+)) bearing markers from two different exogenous DNA species was also detected 15 min after exposure of ind his recipient protoplasts to a mixture of ind(+)his and ind his(+) donor DNA molecules. The unity of the recombinant molecule was ascertained by dilution experiments and by its being resistant to ribonuclease and trypsin treatment (but being sensitive to deoxyribonuclease). The formation of recombinant molecules showed an inverse kinetics to that of the intracellularly induced loss of linkage between the corresponding markers in the wild-type DNA, thus suggesting a breakage and reunion process which is also favored by the absence of DNA synthesis in the protoplasts and the effect of some specific inhibitors.     

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

The Size and Continuity of DNA Segments Integrated in Bacillus Transformation

We investigated the size and continuity of DNA segments integrated in Bacillus subtilis transformation. We transformed B. subtilis strain 1A2 toward rifampicin resistance (coded by rpoB) with genomic DNA and with a PCR-amplified 3.4-kb segment of the rpoB gene from several donors. Restriction analysis showed that smaller lengths of donor DNA integrated into the chromosome with transformation by PCR-amplified DNA than by genomic DNA. Nevertheless, integration of very short segments (<2 kb) from large, genomic donor molecules was not a rare event. With PCR-amplified segments as donor DNA, smaller fragments were integrated when there was greater sequence divergence between donor and recipient. There was a large stochastic component to the pattern of recombination. We detected discontinuity in the integration of donor segments within the rpoB gene, probably due to multiple integration events involving a single donor molecule. The transfer of adaptations across Bacillus species may be facilitated by the small sizes of DNA segments integrated in transformation.     

Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid transport in membrane vesicles from Bacillus subtilis.

The rate of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane vesicles from Bacillus subtilis W23 increases three- to fourfold during logarithmic growth, reaching maximal levels in early stationary phase. Initial rates of L-proline, L-alanine, and L-glutamate transport energized by NADH closely parallel the increase in NADH oxidation. In vesicles prepared at different stages of growth, a constant number of NADH molecules varying from 150 to 260 have to be oxidized to transport one molecule of amino acid. Membrane vesicles from B. subtilis aroD (strain RB163), a mutant defective in menaquinone synthesis, do not transport amino acids in the presence of NADH. Ascorbate plus phenazine methosulfate, however, energizes amino acid transport equally well as in vesicles of B. subtilis W23. NADH oxidation and NADH-driven amino acid transport can be restored instantaneously by the addition of menadione (vitamin K3).     

Nutritional Factors Influencing the Development of Competence in the Bacillus subtilis Transformation System

Cultures of Bacillus subtilis developed competence for the uptake of deoxyribonucleic acid in a chemically defined medium with a predictable, reproducible pattern. The gross effects of individual amino acids were determined. Seven amino acids, most of which are reported to be major components of the cell wall, were shown to impair the development of maximal levels of competence. When the synthetic growth medium was supplemented with a mixture of the nine amino acids which we found to stimulate the development of competence, the level of transfection was increased to 10 to 15% of the population. The actual level of competence in these populations was assayed by transformation of unlinked bacterial markers and by two different transfection assays. The results indicate that calculations from cotransfer of unlinked markers overestimates the degree of competence in highly competent populations of B. subtilis, whereas the number of plaques obtained in transfection is an under-estimate of the actual level of competence. The results are interpreted to indicate that neither method of analysis gives a true estimate of the competent population, but that more than 80% of the cells may be competent.     

Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis

This paper describes the major transmutations of donor deoxyribonucleic acid (DNA) after uptake by competent Bacillus subtilis cells. Kinetic experiments confirm that after exposure to competent cells, donor DNA is converted to double-stranded fragments (DSF) which can be isolated as early as 30 s from the beginning of the reaction. At this time, DSF represent the only identifiable product of donor origin. After 1 to 2 min, DSF are converted to deoxyribonuclease-resistant forms, identified as single-stranded DNA fragments (SSF). SSF are intermediates in the transformation process leading to the formation of donor-recipient complex. This component makes its appearance between 2 to 4 min from the beginning of the transformation process. All the donor-recipient complexes found at the end of the reaction can be accounted for quantitatively by the DSF and the SSF found in the initial stages of transformation. A quantitative discussion of the transformation process is included.     

Restriction and modification in Bacillus subtilis: effects on transfection under marker rescue conditions

The role of homology between donor and recipient DNAs in the protection of transfecting DNA against restriction by competent Bacillus subtilis R cells was studied under marker rescue conditions with modified helper phage. By comparing restriction under conditions of preinfection marker rescue and superinfection marker rescue, the significance of DNA homology during the initial stages of DNA processing by competent cells could be studied. The results showed that both in preinfection and in superinfection, complete protection against restriction of transfectants produced via rescue by the modified homologous helper chromosome occurred. Even up to 90 min after entry, DNA entering the helper-mediated pathway of transfection was not affected by restriction. The significance of these findings is discussed in the general context of the role of DNA homology between donor and recipient on the fate of donor DNA in competent B. subtilis, in particular in relation to the effects on restriction.     

Effect of pancreatic DNAse on Bacillus subtilis multiplication depending on the physiological state of the culture

The effect of pancreatic DNAase on the growth of Bacillus subtilis, the synthesis of DNA, RNA and protein in the cells was studied as a function of their physiological state. The cells were found to be sensitive to the action of DNAase at the end of the linear growth phase, which coincided with their transition into the competence state and with their ability to adsorb the enzyme on the cell surface.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.     

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.     

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。     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.