D-Glucosamine as inhibitor of early processes of transformation inBacillus subtilis 168trp 2 −

Glucosamine added to a transformation medium (TM2) after a 30-min cultivation of cells exhibited the highest inhibitory effect on the transformation process inBacillus subtilis 168trp ₂ ⁻ . The recipient culture was least sensitive to glucosamine added after 50 min. Glucosamine had no inhibitory effect when added 10 min after the addition of transformation DNA.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

Interspecies transformation in Bacillus: mechanism of heterologous intergenote transformation.

Bacillus subtilis-Bacillus globigii hybrids were made by integration of the B. globigii aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformation by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 X 10(6) to 40 X 10(6) daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigii DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences of both flanks of the intergenote.     

Genetic properties of arsenate sensitive mutants of Bacillus subtilis 168

Summary Arsenate sensitive mutants were isolated from Bacillus subtilis strain 168 after treatment with N-methyl-N-nitro-N-nitrosoguanidine or ethyl methane sulfonate. Though all mutants are phenotypically identical, a high proportion (40%) of the induced mutations are of a multisite nature as they do not revert spontaneously and are poorly transformable to arsenate resistance with wild type DNA. On the basis of transformation efficiency, UV inactivation kinetics and cotransduction frequency of outside markers, four independently isolated multisite arsenate sensitive mutations are characterized as resulting from large deletions of homogenous size (24000±6000 base pairs). The arsenate resistance locus was mapped between phe and aroD on the B. subtilis chromosome by PBS1 mediated transduction. Mechanisms for the formation of such chromosomal deletions are discussed.Part of the dissertation of Alice Adams Lindahl, presented to New York University in partial fulfillment of requirements for the Ph. D. degree. A preliminary report of this work was presented at the NATO International Symposium, Mol, Belgium, August 1970.     

Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194

Summary We have constructed a hybrid plasmid, pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA at its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficieny., as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA proccssing during plasmid transformation of B. subtilis is presented.     

Transformation inBacillus subtilis. Initial stages of the transformation process and their energy dependence

NaN₃ was found to inhibit transformation but not the irreversible binding of donor³H-DNA in competent cells of the original low-transformable strainBacillus subtilis 168trp ₂ . Addition of NaN₃ to cells of two mutantsBacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells. The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN₃, leading preferentially to lysis of the competent cells.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

A very poorly transformable mutation at the threonine deaminase locus in Bacillu subtilis

Summary An isoleucine requiring mutant of Bacillus subtilis which displays some novel properties has been studied. The strain lacks threonine deaminase activity and is also more sensitive than the wild type to the action of mitomycin C without being more sensitive to UV light. The two features (isoleucine requirement and mitomycin sensitivity) are transferred simultaneously in transformation. The isoleucine marker of this strain is situated at the right end in the map of the threonine deaminase locus. It is at present the nearest known marker to the terminus of the B. subtilis chromosome. Relative transformation frequency for this marker is very low suggesting a low integration efficiency. This is true for both alleles (wild type and mutant) of the marker. SP 10 transduction frequency is almost nil whereas PBS-1 transduction is as effective as for any other marker in that region. Any treatment which causes breakage of the strands in wild type DNA (dilution, shearing, deoxyribonuclease I action) brings about a very important decrease in the relative frequency of transformation for this marker. The marker is more sensitive to these treatments than the weak linkage of two very distant markers. Results agree with the hypothesis that the mutation in this strain corresponds to a deletion in the terminal region of the chromosome. The deletion (suggested also by the absence of reversion) should nevertheless be very long, probably exceeding the average length of a transforming DNA segment. However, an alternative hypothesis has been considered and discussed.     

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT¹¹⁰ sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT¹¹⁰ locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT¹¹⁰ and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

Interspecific transformation of rifampicin resistance in the genus Bacillus

Summary Transfer of resistance to rifampicin, a genetic determinant for RNA polymerase, was tested by transformation between B. subtilis as recipient and eight other Bacillus species as DNA donors. Seven of these donors effected transformation of rifampicin as well as erythromycin resistance, and cotransfer of these linked markers was also found in the interspecific crosses. The results extend the concept of a region of gene conservation in this genus.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives

An eightfold auxotrophic strain of Bacillus subtilis was constructed. It was about equally well transformable for all markers. When this strain was used as recipient in transformation, single marker transformation frequencies of 0.5–2.0% were obtained. The markers were located relatively to each other using marker frequency analysis. Two UV-sensitive derivatives, equally well transformable as the parental multiple auxotroph, were isolated. One was highly sensitive to UV irradiation, was host cell reactivation-negative and did not show DNA breakdown or recovery of DNA synthesis after exposure to UV. Using UV-inactivated transforming DNA, this strain's transformability was strongly reduced as compared with both the UV-resistant parental strain and the other, moderately UV-sensitive, strain.     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

The fertility of a smooth strain ofEscherichia coli

Auxotrophic mutants of the potentially pathogenic smooth strain ofEscherichia coli O55: B5 were isolated and used as recipients in mating experiments withEscherichia coli K12. The recombination frequency obtained in mating K12HfrH donor with O55 recipient was about 100 to 1,000 times lower than in K12×K12 cross. The F-prime donor O55 strain when backerossed with the K12 recipient again mated with low efficiency. Crosses with derivatives of O55 which were presumed to carry K12 host specificity genes were inconclusive and we cannot exclude the possibility that O55 restricts K12 DNA. A polyauxotrophic mutant of O55 with better mating ability was isolated. The improved genetic homology in K12 and O55 chromosomes could account for increased frequency of recombinants obtained. This strain was used to establish the location of the loci for O55 and B5 antigens. No close linkage was found between these two antigenic determinants, locus for O55 antigen being located close tohis marker and locus for B5 antigen rather totrp marker.     

An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout

We provide a simple but very efficient transconjugation protocol for Bacillus megaterium. By combining utile attributes of known transconjugation methods (small size of the transferred DNA, close physical contact between donor and recipient cells, and heat treatment of the latter) and by determining the appropriate donor/recipient ratio, mating approaches yielded 5 × 10⁻⁵ transconjugants/recipient. Counter-selection for eliminating Escherichia coli donor cells from the mating mixture was possible by pasteurization in case a wild type sporulation proficient B. megaterium served as the mating partner. For nonsporulating mutants, the sacB gene from Bacillus subtilis coding for levansucrase was successfully employed to select against the E. coli donor. The transfer efficiency, up to 15,000 transconjugants acquirable in a single experiment, sufficed—for the first time in this species—to directly select a gene (uvrA) knockout in a one-step procedure. By making use of a mobilizable B. megaterium suicide vector, ten out of the 40 sampled putative transconjugants displayed the expected UV sensitivity and were found to harbor the suicide vector at the anticipated position. Along with the soon available information arising from current B. megaterium sequencing projects, the possibility to quickly inactivate genetic loci will considerably speed up genetic work with this biotechnologically relevant species.     

Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts

Summary Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.     

Molecular heterozygotes in Bacillus subtilis and their correction

Summary Data are presented on the probability of correction of molecular heterozygotes during the transformation of Bacillus subtilis. This value varies between 0 and 1 for different mutants in the same genetic locus. The correction of closely linked markers is simultaneous but it is independent for distant loci. The efficiency of integration of different genetic markers during transformation depends on their ability to be corrected towards the structure of the recipient strain.The linked correction of neighboring markers explains quantitatively the asymmetric phenomena in reciprocal crosses.UV-irradiation of transforming DNA inhibits strongly the correction of molecular heterozygotes and eliminates the asymmetry in reciprocal crosses. The same effect is found after chemical damage of DNA by nitrous acid.     

Simple Measurement of Blood NADP and Blood Levels of NAD and NADP in Humans

The tryptophan synthase genes, trpA and trpB, of Bacillus stearothermophilus IFO13737 were cloned by transformation of tryptophan auxotrophic mutations of the trp genes into Escherichia coli. The genes are located in the order of trpB and trp A, according to their coding orientation, in a 2.5 kb EcoRy-Hindlll DNA fragment. The complete nucleotide sequence of this DNA was determined. The trp A and trpB genes consist of 810bp (269 amino acid residues) and 1215bp (404 amino acid residues), respectively. The 5′-proximal portion of the trpB gene was found to overlap 20 nucleotides of the upstream coding region of the trpA gene. The homology of the amino acid sequences of the trp gene products of trp A and trpB of B. stearothermophilus is 35 and 50 %, respectively, to those of E. coli, and 55 and 70 %, respectively, to those of B. subtilis.