Comparison of Deoxyribonucleic Acid Uptake and Marker Integration in Bacilli and Protoplasts of Bacillus subtilis

trp(+)his(-) donor deoxyribonucleic acid (DNA) was added to highly competent trp(-)his(+) recipient bacilli and to protoplasts prepared from these bacilli, and the cell-DNA complexes were incubated for 30 min. The complexes were then washed and lysed, and their DNA was analyzed on a trp(-)his(-) strain for the donor marker trp(+), the resident marker his(+), and for the recombinant trp(+)his(+) combination. The extracts of the bacillary complexes contained a normal percentage of donor markers (0.1-0.2%), and the number of trp(+)his(+) doubles (20% of all trp(+) transformants) indicated that the donor DNA had become integrated into the resident genomes. The protoplast complexes contained 10 to 1,000 times fewer donor markers and almost no recombinants. This indicated that, in protoplasts, marker uptake was minimal and recombination was absent. Uptake was also measured with (3)H-labeled DNA. On the average, protoplasts took up one-fiftieth as much DNA as bacilli. It was concluded that, probably, protoplasts took up no DNA at all, that there were no DNA affinity sites on the surface of the protoplasts, and that the residual marker and radioactivity uptake was due to imperfections in the experimental system. The data and conclusions differed sharply from earlier ones of Hirokawa and Ikeda despite the fact that the techniques of these authors were followed in repeat experiments.     

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.     

Deoxyribonucleic Acid-Membrane Complexes in the Bacillus subtilis Transformation System

The fate of transforming deoxyribonucleic acid (DNA) in Bacillus subtilis was studied by isolating the DNA-membrane complex on Renografin gradients. Soon after uptake, transforming DNA binds to the cell membrane and displays a greater resistance to shear than the recipient genome-membrane complex. Ten minutes after uptake, a portion of the donor DNA is released from the membrane. Most of the released donor radioactivity represents unintegrated, biologically inactive DNA. Recombinant or integrated DNA is enriched 1.5- to 1.7-fold in the membrane. This enrichment last at least 30 min after termination of DNA uptake, and probably much longer. The data suggest that transforming DNA may be integrated into the recipient genome on, or close to, the cell membrane.     

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.     

Chromatographically Fractionated Complementary Strands of Bacillus subtilis Deoxyribonucleic Acid: Transformation of Hybrids

The annealing properties as measured by the restoration of transforming activity and hypochromicity of methylated albumin-kieselguhr (MAK)-fractionated complementary strands of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. Temperature-absorbance measurements performed on annealed mixtures of various L and H strand fractions indicated the existence of a complementarity gradient between the two MAK peaks. The markers purA16, leu-8, metB(5), thr-5, and the linked marker hisB(2)-try-2 exhibited different bimodal distributions on MAK columns. The transforming efficiency of heteroduplex mixtures, prepared by cross-annealing resolved complementary strands of wild-type and recipient DNA, was compared. The transforming efficiency of the wild-type L and H strands was equal in one preparation and unequal in a second preparation. It was found that in the second strand preparation the heteroduplex DNA containing the H strand from wild type was more efficient for all of the markers tested. The variations in transforming efficiencies of the complementary strands in heteroduplex molecules reported here and by others are due in part to strands of unequal length and probably to the self-annealing property of the H strands. At present, no conclusion could be made regarding the existence of strand selection bias during integration of donor DNA in competent B. subtilis cells.     

Genetic fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation.

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.     

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

Repair of ultraviolet-irradiated transforming deoxyriboinucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation. Two mutations that cause a Uvr- and phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA. Genetic and kinetic evidence is presented to show that integrated DNA was apparently repaired by both excision and postreplication repair in wild-type and in uvr-1 recipients, although the latter excise pyrimidine dimers very slowly. In uvr-42 mutants, which are defective in incision at pyrimidine dimers, dimer-containing DNA was integrated. Postreplication repair apparently saved uvr-42 recipient cells from the lethal effects of integrated dimers, but the recombination events accompanying postreplication repair greatly reduced the linkage between closely linked genetic markers in the donor DNA. Repair of transforming DNA in a recG recipient, which does excision repair but not postreplication repair, was nearly as efficient as in wild-type cells. However, in this recipient linkage was altered only slightly, if at all, compared with wild-type cells. The apparent reduction in size of integrated regions of ultraviolet-irradiation transforming DNA probably results mainly from postreplication repair of larger integrated regions.     

Thymidylate synthesis and aminopterin resistance in Bacillus subtilis

Wilson, Melba Carr (Brown University, Providence, R.I.), James L. Farmer, and Frank Rothman. Thymidylate synthesis and aminopterin resistance in Bacillus subtilis. J. Bacteriol. 92:186-196. 1966.-The thymine-requirement of Bacillus subtilis 168 thy results from mutation in two unlinked genes (i.e., genetic loci) designated thyA and thyB. The thyB gene is located between the met and ile markers. Both thyA(+)thyB and thyA thyB(+) strains are phenotypically thy(+). ThyA(+)thyB strains resemble the wild type in their sensitivity to aminopterin, poor incorporation of exogenous thymine into deoxyribonucleic acid (DNA), and high level of thymidylate synthetase activity in crude extracts. ThyA thyB(+) strains are resistant to aminopterin in the presence of thymine, incorporate exogenous thymine into DNA, and have no detectable thymidylate synthetase activity. Experiments designed to elucidate the role of the thyB gene indicate that it specifies an alternate pathway of thymidylate synthesis, similar to thymidylate synthetase but requiring a cofactor other than tetrahydrofolate. The mechanism of selection of thymine-requiring mutants by aminopterin is revealed by these results.     

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com⁻⁵⁶, was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.     

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.     

Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.     

Intergenotic Transformation of the Bacillus subtilis Genospecies

A multiple auxotrophic derivative of Bacillus subtilis 168 (strain BR151 carrying lys-3, trpC2, metB10) was transformed with deoxyribonucleic acid (DNA) isolated from B. subtilis 168, Bacillus amyloliquefaciens H, B. subtilis HSR, Bacillus pumilus, and Bacillus licheniformis. Transformation with heterologous DNA occurred at a very low frequency for the three auxotrophic markers. Heterologous transformation to rifampin resistance was 100 to 1,000 times more efficient than transformation to prototrophy. Transformants from the various heterologous exchanges were used to prepare donor DNA. The fragment of integrated DNA from the heterologous (foreign) species, termed the "intergenote," was capable of transforming BR151 with an efficiency almost equal to that of homologous DNA. When BR151 DNA contained the Rfm(R) (rifampin resistance) intergenote from B. amyloliquefaciens H, the frequency of transformation was frequently greater than that of the homologous DNA. Accompanying this increased efficiency was a marked change in the physiology of the cells. The growth rate of the transformants carrying this intergenote was approximately one-half that of either parental strain. Thus, in a prokaryotic transformation system, adverse side effects can occur after incorporation of a segment of foreign DNA.     

Fate of Transforming Deoxyribonucleic Acid After Uptake by Competent Bacillus subtilis: Nonrequirement of Deoxyribonucleic Acid Replication for Uptake and Integration of Transforming Deoxyribonucleic Acid

Studies on transformation of Bacillus subtilis using the inhibitor 6-(p-hydroxyphenylazo)-uracil show that deoxyribonucleic acid (DNA) replication is not required for the uptake and integration of donor DNA and genetic markers.     

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.     

Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae

Voll, Mary Jane (University of Pennsylvania, Philadelphia), and Sol H. Goodgal. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883. 1965.-Transforming deoxyribonucleic acid (DNA) which has been irreversibly removed from solution by competent cells undergoes a progressive loss in marker activity when tested by lysis of the cells and exposure to new recipient cells. The loss of activity is limited and marker-specific, with greater inactivation of those markers with lower efficiencies of transformation. Recipient factors or donor factors which have undergone recombination, as measured by the appearance of linked markers, do not undergo inactivation. The efficiency of transformation can be correlated with the sensitivity of a marker to inactivation after DNA uptake. A mutation which affects the efficiency of transformation is found to increase sensitivity to postuptake inactivation. The rate of inactivation is temperature-dependent. At temperatures of 20 and 45 C, marker inactivation can occur without concomitant recombination. During the uptake process, DNA is retained in an acid-insoluble form, indicating that the fate of Haemophilus influenzae DNA differs from the fate of transforming DNA in pneumococcus.     

Isolation of Bacillus subtilis genes from a charon 4A library.

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.     

Spontaneous transformation characteristics of Bacillus subtilis bacteria. I. The relatively low transforming activity of spontaneously released DNA for markers located close to the origin and termination points of chromosome replication]

Relative efficiencies of spontaneous Bacillus subtilis transformation for markers placed in different areas of the cell chromosome were studied. As donor of genetic material, an untransformable strain BD224 trpC2 thr5 rec4 was used during its early log-phase. It was found that for markers placed near points of origin and termination of the chromosome replication the relative transformation efficiencies are significantly lower than those in the case of transformation with DNA extracted from the same donor cells. If a contact of spontaneously released DNA with the recipient cells was delayed for about 60 minutes ("separate" experiment) this difference proved to be less pronounced for ade16 placed near the origin, but remained practically unchanged for metB placed near the termination point. The results obtained can be explained by permanent attachment of chromosome regions, carrying "origin" and "termination" points, to a cytoplasmic membrane. During spontaneously release of cellular genetic material, "origin" and "terminal" DNA fragments carrying ade16 and metB respectively, can retain the contact with components of cell membrane. Hence, their penetration in the recipient cell and (or) participation in recombination can be violated. The first of two fragments becomes free from structurating substances more easy.     

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.     

Genetic Studies Relating to the Production of Transformed Clones Diploid in the Tryptophan Region of the Bacillus subtilis Genome

Transformation experiments with Bacillus subtilis strains carrying trpE26 (the marker responsible for the detection of merodiploid clones after transformation or transduction) have established the precise position of this marker on the "aromatic region" of the chromosome, at the distal end of the anthranilate synthetase locus. Integration efficiency of the mutant allele (trpE26) seems to be very low. Co-transfer of markers situated on either side of it is almost nil when both donor and recipient carry this mutation. The "exclusion" of trpE26 does not, however, affect recombination frequencies for nearby markers. To explain these facts we considered the hypothesis of a preferential breakage of the deoxyribonucleic acid (DNA) at the trpE26 site or that of an insertion mutation. These studies have also demonstrated the establishment of physical linkage of a marker from the exogenote (hisH2) to a resident marker (tyrA1) in stable and unstable merodiploid clones, thus confirming integration of the donor DNA segment into a genetic structure of the recipient. Furthermore, duplication was shown in merodiploid clones (through reversion and transformation) for a locus of the recipient (tyrA) which was not involved in the initial transformation. This suggests that the diploid condition in this region extends beyond the transformed area. Interpretation of the genetic constitution of these partial diploids calls for postulation of the existence of long duplications, a second (incomplete) chromosome, or an episome-like element.     

DNA Repair and the Evolution of Transformation in Bacillus Subtilis. III. Sex with Damaged DNA

Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. We document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome.