Colony mutants of compatible nocardiae displaying variations in recombining capacity.

Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

Linkage and segregation of unselected markers in matings of Nocardia erythropolis with Nocardia canicruria.

The segregation of unselected genes expressing resistance or susceptibility to acriflavine, erythromycin, streptomycin, and tetracycline was analyzed in selected prototrophic recombinants resulting from matings of Nocardia erythropolis and N. canicruria. The organisms were shown to be functionally haploid and appeared to contain not more than one genome. It was postulated that all observed genes were present in a linear linkage group. The ordering of the genes in N. erythropolis was: tetB10 eryB9 his-3 purA1 acr-2 strA1 (respectively, resistance to tetracycline and erythromycin, deficiency for histidine and for purine, and resistance to acriflavine and streptomycin). The ordering of the genes in N. canicruria was: purB2 tetA9 eryA7 acr-11 strB2 (respectively, deficiency for purine, and resistance to tetracycline, erythromycin, acriflavine, and streptomycin). Excluding the genes for acriflavine resistance, acr-2 and acr-11, resistance loci in N. erythropolis were not allelic to and showed lateral displacement from genes controlling phenotypically similar resistance in N. canicruria. Evidence for some lack of homology between N. erythropolis and N. canicruria genomes was found. Recombination phenomena between the nocardial species was postulated to occur as a result of formation of a heterogenomic zygote in which new combinations were produced. Production of selectable, haploid recombinants was ascribed to subsequent haploidization of the zygote.

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.

Inheritance of mating factors in nocardial recombinants.

The segregation of mating loci with other unselected genes was analyzed in recombinants obtained from matings of Nocardia canicruria and N. erythropolis. The loci C/c and E/e control nocardial compatibility. Four mating genotype combinations were observed: cE, Ce, CE, and ce. Strains of N. erythropolis bear the genotype cE and strains of N. canicruria bear the Ce alleles. The CE recombinant mating type is capable of mating with both organisms, whereas the ce-containing recombinant is nonfertile. The E locus was found to segregate with StrA1 (streptomycin-resistance gene) on the N. erythropolis linkage group. The C locus appeared linked to PurB2 (purine-requiring gene) on the N. canicruria linkage group. A few observed recombinants were capable of further segregation of unselected characters after colonial purification, suggesting a possible heterogenomic condition or multiple rounds of mating. Prior treatment of recombinants with acriflavine failed to alter their compatibility or the frequency at which recombinants were recovered. The segregation pattern of the mating loci allowed for specific recombinant class types to be compatible.

Inactivation of nocardiophages phi C and phi EC by extracts of bacteriophage-attachable cells.

Cultures of several species of Nocardia, including N. erythropolis Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages phiC and phiEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages phiC and phiEC were inactivated by the same complex. However, phage phiEC inactivation was 10-fold greater than phiC inactivation. The velocity of inactivation was about 4.1 x 10(2) plaque-forming units per microgram per minute for phiC and 1.1 x 10(3) plaque-forming units per microgram per minute for phage phiEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.

Heterogenomic recombinants from compatible nocardiae.

Recombinants obtained from matings of Nocardia erythropolis x N. canicruria were tested for their genetic stability by comparing phenotypes from direct selection with the same population after unselected growth. Contraselective loci were employed in various combinations in order that all of the mapped characters might be subjected to unselected analysis. Some recombinant class types appeared as stable haploids, whereas others behaved as heterozygous diploids, segregating out new phenotypes. All regions of the parental genomes were found to be involved in segregation, implying that the entire mapped region can become merozygotic under standard mating conditions. On the basis of segregating phenotypes, the genetic potentials of these compatible nocardiae were ascertained as follows: the formation of a diploid with subsequent segregation of parental or haploid recombinant genomes or both; persistence of the diploid through many generations; continuing reassortment of genetic information by multiple matings between parental or recombinant organisms; and, very probably, second-round recombinations within the diploid. A considerable difference in the nuclear division time between the parental organisms was postulated to have significant effects on the nature of the unselected segregants.

Isolation and characterization of a lysogenic strain of Nocardia erythropolis.

A stable phage-carrying strain of Nocardia erythropolis was isolated from an infection with the nocardiophage phiEC. Growth of the strain in phage-specific antiserum for 48 hr produced cured organisms at a frequency of about 0.5%. Spontaneous curing, determined by serial single-colony isolations, was less than 0.4%. The strain could not be infected by phage phiEC nor by a closely related phage, phiC, although the cells were able to adsorb these phages. In cell populations, a frequency of 2.5 x 10(-4) cells spontaneously induced. The growth rate of the strain was comparable to that of the uninfected wild-type N. erythropolis. Ultraviolet irradiation or treatment with mitomycin C induced the strain to produce larger numbers of phage. It was concluded that the isolated strain was lysogenic.

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.

The recovery of nocardial recombinants from broth cultures.

Broth culture media were examined for their ability to support growth and recombination between compatible strains of Nocardia erythropolis. Nutrient(Nut) and peptone-yeast-extract (PY) broths supported the production of recombinants after 36 h of incubation with a maximum recovery of about 6.0 times 10(-7) CFU/ml. Cells mated in trypticase broth (TB) yielded the highest incidence of recombinants (1.0 times 10(-2) CFU/ml) in the absence of parental cell growth. From a chemically defined mating broth (CD), supplemented with limited amounts of the parental-growth requirements, recombinant recovery reached about 1.0 times 10(-4) after 120 h of incubation. The recombinant class types obtained from Nut- or PT-mated strains were predominantly auxotrophic while TB-mated strains produced stable proteotrophs. The high incidence of recombinant types from TB-mated strains was due to growth of selected prototrophic classes. Studies with strains mated in PY broth indicated that the mating event occurs at very low frequencies between older, stationary-phase cells rather than between actively growing, log-phase cells.

Genophore homologies among compatible nocardiae.

Deoxyribonucleic acid (DNA) reassociation analyses were employed to determine the molecular relationships between recombinable nocardiae. Analysis of the compatibility system of Nocardia erythropolis Mat-Ce and Mat-cE mating strains demonstrated the existence of extensive homology under both exacting and nonexacting conditions. Labeled N. erythropolis Mat-cE DNA reassociated equally as well with the Mat-Ce test DNA as with its own filter-bound DNA. However, the Mat-cE DNA bound only ca. 60% of the Mat-Ce DNA, when the latter was the reference. The existence of unique nucleotide sequences is postulated on the basis of these results as well as of aberrant segregation patterns which have been observed in certain class types of recombinants. Reassociation data reveal that recombinants representing the inheritance of different portions of each of the parental genomes have inherited the unique portion from the Mat-Ce parent. N. restrictus AY-B-226 exhibited little relatedness (11 to 32%), and N. globerula ATCC 9356 only slightly more (21 to 42%), to either of these mating strains at either exacting or nonexacting temperatures of incubation.

Inheritance of nocardiophage phi EC in matings of nocardial lysogens.

Lysogens of Nocardia erythropolis were mated with nonlysogenic strains to study the inheritance of the phi EC prophage. Crosses between lysogenic strains of the Mat-Ce mating type and nonlysogenic Mat-cE strains produced Mat-cE lysogens at a recovery rate of 17%, whereas recombination frequencies between chromosomal traits were about 2.3 x 10(-5). Crosses of lysogenic Mat-cE mating types with nonlysogenic Mat-Ce produced Mat-Ce lysogens at a recovery rate of 19%, whereas recombinants for chromosomal traits were recovered at only 1.8 x 10(-5). Crosses of homologous mating types, lysogenic Mat-Ce with nonlysogenic Mat-Ce or lysogenic Mat-cE with nonlysogenic Mat-cE, failed to transfer the prophage. It was concluded that the phi EC prophage exists as a plasmid and can be transferred at high frequencies with patterns of transfer controlled like typical nocardial fertility. Evidence that the prophage may also exist as an integrated element was observed from recombination analyses.

Aminoglycoside modification by gentamicin-resistant isolates of Staphylococcus aureus.

Three clinical isolates of Staphylococcus aureus, which were previously shown to contain a 50S plasmid conferring resistance to several aminoglycosides, were examined for modifying enzymes. Both the wild-type and heat-cured derivatives of the isolates were screened for acetyl-, adenylyl-, and phosphotransferase activities. The substrates were gentamicin, amikacin, and netilmicin; the results indicated that even though all three activites were present, the phosphotransferase reaction was most responsible for resistance to these antibiotics. The absence of any of the modifying activites in cured derivatives of the three isolates supports the conclusion that aminoglycoside resistance in these strains is conferred by a plasmid.