Quantitive Autoradiographic Study of Competence and Deoxyribonucleic Acid Incorporation in Bacillus subtilis

The relationships among deoxyribonucleic acid (DNA) uptake, transformation, and autoradiographic labeling were investigated. It is shown that: (i) autoradiography is a good method for measuring the total fraction of competent cells able to incorporate transforming DNA; in our best experiment that fraction was 11.5%. (ii) Computation of the fraction of competent cells in a culture of Bacillus subtilis, by comparing the frequencies of single and double transformants for two unlinked markers, gives values which are somewhat different from those obtained by auto-radiography. (iii) On the average, a competent cell of B. subtilis irreversibly takes up from 0.9 x 10(-10) to 1.4 x 10(-10) mug of DNA; this amount represents about 1/55 to 1/35 of the bacterial chromosome weighing 5 x 10(-9) mug, and corresponds to three to six DNA molecules having a molecular weight of 1.6 x 10(7).     

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

The chromosomes of a tryptophan(-), thymine(-) double auxotroph of Bacillus subtilis were uniformly aligned at the chromosome terminus by an amino acid starvation treatment. By subsequent incubations, the starved culture was rendered competent, while its state of synchronous chromosome arrest was maintained by thymine starvation. The competent, chromosome-arrested cells were transformed for three unlinked markers, located in two different chromosome regions. Shortly after addition of deoxyribonucleic acid, the cell walls were removed with lysozyme in a medium containing deoxyribonuclease and no thymine, and the protoplasted culture was assayed for single and double transformants. It was found that markers both near and distant from the terminus entered freely into the cell interior. There was no important difference in the relative frequency of entry of different markers between synchronously arrested cells and nonsynchronized control cultures. It is concluded that entry of a given marker into the cell interior can occur even if the replication site of the chromosome is stationary at a location distant from the locus of the resident homolog of the entering marker. A mechanism of donor deoxyribonucleic acid entry involving homology at the replication fork is excluded.     

Number of Transformable Units Per Cell in Diplococcus pneumoniae

Analysis of frequencies of single and random multiple transformations in Diplococcus pneumoniae showed that there are at least two transformable units per cell of the total population in highly competent cultures. If 100% of the cells are competent in these cases, the units may be interpreted as the strands of one duplex deoxyribonucleic acid recipient chromosome. The theory is developed to allow for extension to more complex situations.     

A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis.

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.     

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

Tryptophan- and thymine-requiring cells of Bacillus subtilis, emerging from an amino acid starvation treatment which causes arrest of the chromosomes at the terminus, were not transformable. During subsequent incubation in a thymineless medium supplemented with amino acids, the cultures developed competence while retaining chromosome arrest. The competent subpopulation apparently shares the synchronous chromosome arrest of the bulk population. This was shown by different methods. The principal method was marker frequency analysis of the deoxyribonucleic acid extracted from a population enriched for competent cells by a column-chromatographic method. It is concluded that development of the competent state can occur in nondividing cells, and that the presence of a replication fork actively engaged in synthesis of deoxyribonucleic acid is not required for the development of this state.     

Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae.

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.     

Competence and Deoxyribonucleic Acid Uptake in Bacillus subtilis

The distribution of uninucleate and multinucleate cells of Bacillus subtilis, fractionated by zonal centrifugation, shows that the uninucleate cells are most likely to be competent. Only the competent fraction of the population incorporates deoxyribonucleic acid.     

Different nuclease activities in competent and noncompetent Bacillus subtilis.

Competent and noncompetent cells of Bacillus subtilis were separated on the basis of their different buoyant densities. The two types of cells were compared with respect to their interactions with exogenous deoxyribonucleic acid(DNA). After exposure of DNA to the cells, the unadsorbed fraction of DNA molecules was examined. Both types of cells decreased the biological activity of this DNA, the inactiviation exerted by noncompetent cells being more severe than that exerted by competent cells. Sedimentation analysis of the inactivated DNA revealed that fragments of DNA are produced, owing mainly to the introduction of double-strand scissions. In addition to this fragmentation, the competent bacteria extensively digested the DNA exonucleolytically. This type of breakdown was specifically related to the competent state rather than to the state of low density. The exonucleolytic activity is, in all probability, associated with the cell envelope, because most of the activity is released into the medium when the cells are converted to protoplasts. At 37 C the competence-specific exonucleolytic breakdown started 2 to 3 min after the binding of DNA to the cells. In unfractionated cultures, breakdown may proceed until 70% of the total amount of DNA added has been made acid soluble. Nontransforming Escherichia coli DNA was also subject to exonucleolytic degradation; it seems unlikely,therefore, that this type of breakdown occurs as a consequence of recombination. Since ethylenediaminetetraacetate blocked both transformation by native DNA and the exonucleolytic breakdown of bound DNA, we suggest that the breakdown of DNA by competent cells fulfills an essential function in genetic transformation of B. subtilis.     

Thyroid hormone directly induces hepatocyte competence for estrogen-dependent vitellogenin synthesis during the metamorphosis of Xenopus laevis

Hepatocytes competent for estrogen-dependent vitellogenin synthesis appeared and increased in number in the liver at the metamorphic climax of Xenopus laevis (A. Kawahara, S. Kohara, Y. Sugimoto, and M. Amano, 1987, Dev. Biol. 122, 139-145). The present study was conducted to determine whether cells competent for vitellogenin synthesis could be induced by thyroid hormone in a primary culture of larval hepatocytes. The thyroid hormone, triiodothyronine (T3), directly induced the competent cells in a primary culture of premetamorphic larval hepatocytes in a dose- and duration-dependent manner. The competency acquired in response to T3 persisted after removal of the hormone. Aphidicholin, an inhibitor of DNA synthesis, failed to block this induction, suggesting the presence of a "precursor cell fraction." This cell fraction in the hepatocyte population increased with the progress of metamorphosis. The thyroid hormone is thus considered the cause of competent cell formation at metamorphic climax.     

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.     

Interactions between exogenous deoxyribonucleic acid and membrane vesicles isolated from competent and noncompetent Bacillus subtilis.

Competent cultures of Bacillus subtilis 168 have been fractionated into a high-competent and a low-competent fraction by a large-scale separation technique. Membrane vesicles isolated from both cell fractions are equally active in the transport of L-glutamate. Both membrane vesicle preparations seem to have similar endo- and exonuclease activities. Also, both preparations are capable of binding deoxyribonucleic acid. However, especially at low deoxyribonucleic acid concentrations (1 mug or less per ml), vesicles obtained from competent cells bind significantly more deoxyribonucleic acid (up to sixfold) than vesicles from noncompetent cells.     

Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Ultrastructure of Competent and Noncompetent Cells and Cellular Changes During Development of Competence

By means of electron microscope autoradiography of component cultures of Bacillus subtilis exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid competent and noncompetent cells can be distinguished. Competence is not limited to a specific phase of the cell division cycle. With serial section electron microscopy of competent and noncompetent cells, two types of mesosomal structures are observed: mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and mesosomes which are additionally connected to the nuclear bodies (nuclear mesosomes). The two types show different cellular distributions. Especially the number of nuclear mesosomes is higher in competent than in noncompetent cells. This, and the observation that the increase and decrease of competence is correlated with both the number of cells carrying nuclear mesosomes and the number of nuclear mesosomes per cell, suggests that mesosomes are involved in the acquisition of competence.     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

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.     

The effect of penicillin on competence in Bacillus subtilis cultures growing in chemostat at different doubling times

Summary The relationships between penicillin-resistance and competence at different doubling-times have been studied in a chemostat. Both subinhibitory and lethal levels of drug have a differential effect on competent fraction and cell viability. Transformation frequency was rather increased in the presence of inhibitory doses of penicillin when partially affected cells were protected from lysis by using an isotonic medium; however, transformation frequency decreases when protoplasts were detected.The influence of the doubling-time in the ability of the recipient population to undergo transformation was confirmed. Our results, in accordance with that previously found, might suggest that competence in log phase and competence in the stationary phase could differ in some important aspects.