Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study.

Combined genetic transformation and nutritional assay for identification of Moraxella nonliquefaciens.

A combined genetic transformation and nutritional assay is described that permits definitive identification of clinically isolated strains of Moraxella nonliquefaciens. Crude DNA preparations of strains of various Moraxella species were used to transform nutritional mutants of a stably competent strain of M. nonliquefaciens for ability to grow on a defined medium (Mn-B). DNA samples from 24 independently isolated strains of M. nonliquefaciens all resulted in massive (4+) transformation of each of two mutant assay strains. DNA samples from strains of M. bovis and M. lacunata frequently gave weak (1+) transformation of one of the mutant assay strains (Mn64) but almost always failed to transform another assay strain (Mn136). DNA samples from eight other Moraxella species failed completely to transform either of the mutant assay strains. When streaked on the defined medium used for the transformation assay (Mn-B), 23 of the 24 strains of M. nonliquefaciens grew well, but all strains of M. bovis and M. lacunata failed to grow on this medium.

Defined medium for Moraxella bovis.

A defined medium (medium MB) for Moraxella bovis was formulated. Nineteen strains grew well on medium MB. One strain was auxotrophic for asparagine, and another was auxotrophic for methionine. Strains of M. equi and M. lacunata also grew on medium MB. All strains had an absolute requirement for thiamine and were stimulated by or actually required the other growth factors in the medium.

Defined medium for Moraxella (Branhamella) catarrhalis.

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.

General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens.

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.

Transformation assay for identification of psychrotrophic achromobacters.

The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototrophy. Genetically interacting bacteria included strains that lacked one or more of the characteristics typical for most achromobacters. An oxidase-negative mutant of one of these strains reacted positively in the transformation assay, unlike other oxidase-negative bacteria. Achromobacters were derived from fish, poultry, irradiated foods, seawater, and other sources. One strain previously classified as Micrococcus cryophilus has been shown to be related to the achromobacters. Two achromobacters had an optimum growth temperature of 35 degrees C and behaved as typical mesophiles. The moraxellae and Acinetobacter were shown to be unrelated to the achromobacters by using the transformation assay. The ready demonstration of genetic relatedness provides a new basis for taxonomic grouping of the psychrotrophic achromobacters.

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 method for distinguishing gonococcal colony types.

Gonococcal colony types can be distinguished by a new procedure that makes use of a dissecting microscope with a concave mirror and a fluorescent lamp. Critical adjustment of the mirror angle results in illumination similar to that obtained in the dark-field microscope. When the concave mirror is set at a certain angle, colonies of the lenticular types 1 and 2 refract the light coming through them in such a way that an edge of the microscope stage is focused in each colony. By contrast, colonies of types 3 and 4, which are relatively flat, fail to refract incident light. Although distinguishable from each other by differences in color, type 3 and 4 colonies do not display the focusing effect typical for type 1 and 2 colonies and appear uniformly illuminated. This new technique permits the rapid identification and isolation of even a single type 1 or 2 colony in a field of type 3 or 4 colonies, making it possible to obtain and maintain competent colonies (type 1 or 2) for the genetic transformation assay for Neisseria gonorrhoeae strain identification as well as for other purposes.

Genetic Transformation as a tool for detection of Neisseria gonorrhoeae.

A rapid method for the detection of Neisseria gonorrhoeae, making use of the ability of deoxyribonucleic acid samples from clinically isolated strains of this organism to transform nutritional mutants of a particular strain of N. gonorrhoeae, has been described. In addition to using isolated cultures, transforming deoxyribonucleic acid can be obtained directly from the material that adheres to swabs of the cervix or the urethra. The time interval for transfer of swabs to the diagnostic laboratory is not a significant factor. It is not necessary to use pure cultures on primary isolation plates to obtain definitive results. Nongonorrhoeae neisserias, as well as a large variety of commonly encountered unrelated bacteria, do not react or interfere in the transformation assay when using one of the mutant strains under a standardized set of conditions. The entire assay can be completed in less than 24 h. It has also been shown that type T4 cells of the strain of N. gonorrhoeae employed in the present study are competent for genetic transformation, although type T4 cells are transformed at a significantly lower frequency than are type T2 cells of the same strain.