Studies of some naturally occurring auxotrophs of Neisseria gonorrhoeae.

A strain of Neisseria gonorrhoeae requiring arginine, proline, glutamate and cystine as nutritional supplements was transformed, in several steps, to grow in a simple mineral medium containing cystine as the only growth factor with DNA from several clinically isolated strains of this organism. Using DNA from naturally occurring auxotrophs (auxotypes) known to require arginine, hypoxanthine and uracil (Arg-Hyx-Ura-), as well as other factors, it was possible to transfer nutritional markers, one at a time, into such prototrophs to obtain seven single marker auxotrophic strains. Three different uracil markers, two different hypoxanthine markers, an arginine marker, and an isoleucine--valine markers were each introduced into separate strains. Of 114 DNA samples from independently isolated strains of N. gonorrhoeae, 54 were able to transform all seven single marker strains to prototrophy. Six of the single marker strains failed to be transformed to prototrophy by DNA samples from 43 strains, thus demonstrating that all these strains possess at least six nutritional lesions in common. Two strains were shown to contain all seven nutritional lesions, whereas several strains contained some but not all of the seven lesions. Six of the seven single marker strains have been shown to revert spontaneously to prototrophy at low frequencies. During construction of prototrophic strains it was observed that genes conferring sensitivity to growth inhibition by nutrients in complex media were occasionally transferred along with prototrophy.

Simple genetic transformation assay for rapid diagnosis of Moraxella osloensis.

A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis. The transformation assay clearly distinguishes M. osloensis from Acinetobacter. Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis.

Transformation of Acinetobacter calco-aceticus (Bacterium anitratum).

A highly efficient transformation system has been demonstrated in a strain of Acinetobacter calco-aceticus (Bacterium anitratrum). During mixed growth of various stable, unencapsulated, mutant strains, deoxyribonucleic acid (DNA) is liberated and fully encapuslated transformants can be isolated. Purified DNA preparations have been used to transform suitable recipient mutant strains for ability to synthesize capsules, ability to dispense with a growth factor requirement, and resistance to streptomycin. When the wild-type strain is deprived of its capsule, either by mechanical stripping or by mutation, the unencapsulated cells tend to form large clumped masses. A nonclumping mutant of an unencapsulated strain has been isolated. When ability to synthesize capsules is transformed into this nonclumping strain, the resultant cells no longer form chains, unlike the wild-type encapsulated strain. It appears likely that the occurrence of transformation during growth of mixed cultures, with glucose or gluconate as the carbon source, may be the result of osmotic rupture resulting from the inability of unencapsulated strains to oxidize triose phosphates as fast as they are formed. The finding of transformation in Acinetobacter may provide an additional useful organism for the study of this mode of genetic transfer since this strain grows well in a simple mineral medium containing a single oxidizable source of carbon. Furthermore, no special supplementary factors seem to be required for transformation to take place.