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.     

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.     

Evidence for the Translocation of a Chromosome Sement in Bacillus subtilis Strains Carrying the trpE26 Mutation.

The replication order of markers was studied in Bacillus subtilis strains bearing the trpE26 mutation by the use of the density transfer technique. An important difference in this order was observed in comparison with that of strain 168 T-. All markers tested of a chromosome segment extending from trpD to ilvA replicated early, after purB6 and before thr-5. Two markers flanking this region, trpE8 and citK7, replicated late as usual. The results suggested that this segment was shifted in trpE26 strains to a region closer to the origin of replication. PBS-1-mediated transduction crosses corroborated this hypothesis and revealed the position of the translocated segment. (i) Linkage was demonstrated for markers in the segment (hisH2, tryA1, met B3, ilvA2) to thr-5 and ald; (ii) aroB2 and citK7 were found to be linked; and (iii) linkage of cysB3 to thr-5 was lost in trpE26 strains. These findings made it possible to account for the characteristics of the trpE26 mutation and to propose a model explaining the fact that all trpE26+ transformants or transductants are merodiploid. The model calls for fusion of two genetic elements: two independent chromosomes, or two arms of a replicating structure. The resulting chromosome bears a long tandem duplication. Most of the features of this system of merodiploid formation can be interpreted by use of this model: the segregation pattern of the diploids, the stabilization of the unstable clones, and the length of the duplicated region. A relatively stable diploid strain was also studied by the density transfer technique. The data show that it remained diploid for the region corresponding to the translocated segment and are in agreement with the structure predicted by the model.     

Chromosomal Basis of the Merozygosity in a Partially Diploid Mutant of Pneumococcus

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.     

Integration Efficiency in DNA-Induced Transformation of PNEUMOCOCCUS. II. Genetic Studies of Mutant Integrating All the Markers with a High Efficiency

Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.     

Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

One way to do experiments on gene conversion?

Summary Competent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This correction mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.     

Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation

Summary Direct evidence is presented that the mechanism which discriminates against low efficiency markers in transformation of Diplococcus pneumoniae of genotype hex ⁺ acts on them after the formation of donor-recipient heteroduplexes. This conclusion is based on assays of the transforming activity of donor markers in lysates made after various times of incubation of recipient cells following exposure to DNA. The activity of a low efficiency marker rises substantially, indicating formation of native-like heteroduplex structures, and then falls. At 37° C the process is essentially completed 10 minutes after entry, and the apparent half life of a susceptible heteroduplex is 1.5 to 2 minutes. Data from these and other experiments imply that about as many of the surviving low efficiency markers have simply escaped attack as have been inserted into both strands by the excision-repair process suggested by Ephrussi-Taylor.     

Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.     

Mismatch excision and possible polarity effects result in preferred deoxyribonucleic acid strand of integration in pneumococcal transformation.

Heteroduplex deoxyribonucleic acid molecules having a drug resistance marker on one strand and its wild-type allele on the other have been used as donors in pneumococcal transformation. Opposite strands are not equally effective in producing transformants, and this strand bias is not the same, either in direction or magnitude, for various different genetic markers. Selective excision of mismatched base pairs is probably responsible for the large differences in strand efficiency seen with discriminating (hex+) strains, for when the recipient is nondiscriminating (hex-), and therefore presumably lacking an excision enzyme system, strand bias is drastically reduced or altered. The evidence also indicates that excision occurs after integration, as it is provoked by specific donor-recipient mismatch and not by the same mismatch when introduced within donor heteroduplex molecules. Excision can extend to include a neighboring linked marker which would otherwise not be excised, thus altering its intrinsic strand bias as well as its efficiency in transformation. There is a small bias in relative strand efficiency for some markers, not caused by mismatch excision, which perhaps is due to polarity in the integration process itself.     

Isolation of Klebsiella aerogenes mutants cis-dominant for glutamine synthetase expression

We have isolated three strains of Klebsiella aerogenes that failed to show repression of glutamine synthetase even when grown under the most repressing conditions for the wild-type strain. These mutant strains were selected as glutamine-independent derivatives of a strain that is merodiploid for the glnA region and contains a mutated glnF allele. The mutation responsible for the Gln+ phenotype in each strain was tightly linked to glnA, the structural gene for glutamine synthetase, and was dominant to the wild-type allele. These mutations are probably lesions in the control region of the glnA gene, since each mutation was cis-dominant for constitutive expression of the enzyme in hybrid merodiploid strains. Strains harboring this class of mutations were unable to produce a high level of glutamine synthetase unless they also contained an intact glnF gene, and unless cells were grown in derepressing medium. This study supports the idea that the glnA gene is regulated both positively and negatively, and that the deoxyribonucleic acid sites critical for positive control and negative control are functionally distinct.     

Replication terminus of the Bacillus subtilis chromosome.

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.     

Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae

Summary The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect to which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs.We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.     

Genetic Instability in Auxotrophs of SALMONELLA TYPHIMURIUM Requiring Cysteine or Methionine and Resistant to Inhibition by 1,2,4-Triazole

Triazole-resistant (Trz(r)) derivatives of six cysteine- or methionine-requiring (Cym(-)) mutants of Salmonella typhimurium were isolated. Some of the derivatives of each mutant (CTS) were prototrophic, i.e., Cym(-) was suppressed. In every case suppression was initially unstable, Cym(-) auxotrophs being segregated at high frequency, although Trz(r) was stable. After several subcultures on selective medium, CTS strains were classified as either persistently unstable or stabilized. The unstable strains segregated Cym( -) auxotrophs at frequencies of 50-70%, whereas the stabilized strains segregated at frequencies of less than 1%. All suppressed strains had a stable Trz(r) marker co-transducible with cysA. However, there was a correlation between the class of CTS strain and Cym(- ) phenotype. The stabilized strains were Cym(+), whereas the unstable strains were Cym(-). Acriflavin and ethidium bromide increased segregation in the unstable strains, suggesting the involvement of a plasmid. The stabilized strains were refractory to the curing agents. There was no detectable change in the quantity or quality of the S. typhimurium cryptic plasmid. The Trz(r) phenotype of the CTS strains suggested that Trz(r) mutations were of the stable TrzA type. It is suggested that correction of the Cym(-) lesions in CTS strains results from an insertion within the cysCDHIJ gene cluster of a DNA species originating in the cysALKptsHI region of the S. typhimurium chromosome.     

Conjugation transfer of plasmid resistance to gentamycin and other antibiotics in clinical strains of Ps. aeruginosa]

A possibility of conjugation transfer of the markers of the plasmid resistance to gentamicin and other antibiotics from 10 clinical strains of Ps. aeruginosa, isolated from burn patients to the recipient strain of Ps. aeruginosa PTO 629 Rfr was shown. The marker of gentamicin resistance was transferred to 100 out of 110 of the exconjugants, i.e. 86.2 per cent. The rate of the conjugation transfer in the crosses between the clincal strains of Ps. aeruginosa and the recipient strain PTO 629 Rfr with respect to the gentamicin marker was about 10--7. The plasmid resistance markers in the clincal strains Ps. aeruginosa were transferred in various combinations. Transfer of the markers of resistance to streptomycin, carbenicillin, neomycin and combinations Sm, Nm and Sm, Nm, Cm was not achieved.     

A Study of Pneumococcal Merodiploids at the Molecular Level

The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sul^r-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sul ^r-c marker into a stable sulfonamide resistant strain (sul ^r-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd⁺ transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. II. Stimulation of duplication-loss from merodiploids.

Strains of Salmonella typhimurium which contain a duplication of approximately 30% of the genome may be obtained by a simple selective procedure. These strains are highly unstable, losing the duplication when grown on non-selective medium. In this paper we report that treatment of merodiploid bacteria with mutagenic agents stimulates the rate at which haploid segregants are obtained from merodiploid strains. The mutagens which have been tested for this effect are X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), and the azaacridine half-mustard ICR-372.     

The use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains

Summary Methods for the isolation of hybrids in which one or both of the parental strains are industrial yeasts, using mitochondrial mutations as markers for the selection and isolation of the hybrids, are described. The systems used included crosses of industrial strains with auxotrophic laboratory strains which also carried a mitochondrial antibiotic resistance mutation, crosses using an auxotrophic laboratory strain and a petite mutant of an industrial strain carrying a rescuable antibiotic resistance mutation, and crosses using a petite mutant of an industrial strain, carrying a rescuable mitochondrial mutation for antibiotic resistance and a respiratory-competent industrial strain which carried some other marker.     

DNA Fingerprinting and Phylogenetic Analysis of WNIN Rat Strain and Its Obese Mutants Using Microsatellite Markers

Wistar is the oldest rat strain to be introduced in biomedical research, and various stocks of this strain are maintained in laboratories across the globe. The Wistar strain maintained in our facilities is 85 years old and is not typed genetically so far. Recently, two obese mutant rat strains evolved from this stock, one with euglycemia and the other with impaired glucose tolerance. These mutant rat strains, along with the parental Wistar stock and two other rat strains maintained in our facilities (WKY and F-344), were subjected to PCR-based DNA fingerprinting using microsatellite markers to evolve molecular signatures unique to them. Of the 96 markers screened, we identified a marker, leukosianin, that shows polymorphism between the strains tested and thus appears to be quite useful for rat strain identification. Also, the microsatellite data generated were subjected to hierarchical cluster analysis to generate a dendrogram and to estimate the phylogenetic closeness and distance between the rat strains tested. It was observed that the Wistar strain and its mutants maintained in our facility are genetically distinct and phylogenetically separate from the other two standard strains WKY and F-344.