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Resistance factor-mediated streptomycin resistance.

Resistance (R) factor-mediated streptomycin (Sm) resistance differs from classical, high-level, chromosome-borne Sm resistance in its dominance over sensitivity and in the level of its effectiveness (in Escherichia coli approximately 25 mug/ml versus >2,000 mug/ml). In addition, an R factor-containing strain, unlike high-level Sm-resistant bacteria, showed an inoculum effect with respect to its level of Sm resistance. Crude extracts of this strain destroyed the inhibitory activity of Sm and bluensomycin (Blue) on in vitro protein synthesis. The ribosomes from this strain proved to be sensitive to Sm in vitro. The requirements for in vitro inactivation of Sm (and Blue) were determined to be: extract, adenosine triphosphate or deoxyadenosinetriphosphate, and Mg(++). Chromatographic techniques with radioisotopes revealed the formation of an inactivated form of Sm containing adenosine (or deoxyadenosine), phosphate, and Sm in equimolar amounts. The adenylate moiety is coupled to the streptobiosamine residue, rather than to the streptidine ring, of the Sm molecule. The adenylating enzyme, which is not induced by Sm, is located in the periplasmic space of the R factor-containing strain.

Adenine Nucleotides↗

Suppressor system in Bacillus subtilis 168.

Multiple auxotrophic strains of Bacillus subtilis 168 were tested for joint one-step reversion of two or more auxotrophic markers to the wild-type phenotype. Mu8u5u5, a strain requiring leucine, methionine, and threonine, yielded revertants that grew without added methionine or threonine and proved to have a suppressor gene. When transferred by transformation with deoxyribonucleic acid, this suppressor gene also suppressed the adenine mutation in another strain, Mu8u5u6. The one-step double revertants fell into two distinct classes: strains of class su(+) (I) grow well in broth; strains of class su(+) (II) grow poorly. Strains su(+) (II) tend to revert frequently to the su(+) (I) or su(-) state. Conditional lethal mutants of phage phie were isolated which can grow on the su(+) and not on the su(-) strains.

Bacillus subtilis↗

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.

Alleles↗

Sequential entry of transforming markers into Neisseria meningitidis after chromosome alignment.

The kinetics of appearance of transformants as a function of time of exposure to deoxyribonucleic acid (DNA) was examined in Neisseria meningitidis. Incubation with chloramphenicol for as long as 2 hr, which probably leads to chromosome alignment, resulted in augmentation of the lag period before the appearance of the first transformants. The lag periods thus found were dependent upon the marker tested. This permitted the construction of a time map according to the lag periods observed for individual markers. This map was in general agreement with the chromosome map of the recipient strain as determined by marker frequency analysis. Transformation of recipient cells with chromosomes aligned by growth to the stationary phase showed the same type of increased lag in the appearance of transformants before the logarithmic phase of growth had again been reached. These results support the assumption that the nature of the marker accepted by a recipient cell corresponds to the marker present at the replication point of the chromosome. In the absence of DNA and protein synthesis, the uptake of one marker seems to be successively followed by other markers in a linear order determined by the chromosome of the recipient cell.

Chloramphenicol↗

Temperature-sensitive mutants of Dictyostelium discoideum.

Three classes of temperature-sensitive mutants of the cellular slime mold Dictyostelium discoideum have been isolated. One class contains strains able to grow at 22 C but not at 27 C. Cells of these strains can develop into sorocarps at both temperatures. Another class contains strains which can grow at both temperatures but can only develop at the lower temperature. The third class contains strains unable to grow or develop at 27 C. Those strains whose development is temperature-sensitive appear to carry mutations which affect the cells only during the period of aggregation before the construction of a multicellular sorocarp. When pairs of growth-temperature-sensitive (GTS) strains develop in mixed aggregates, temperature-resistant (TR) cells are formed at a frequency of about 10(-4). These TR cells transmit the phenotype in a relatively stable hereditary fashion. However, temperature-sensitive segregants can be isolated from TR strains even after six clonal passages. Mixed incubation of pairs of morphologically aberrant GTS strains was found to give rise to TR progeny which develop normally. These progeny clones independently segregate morphologically aberrant strains and temperature-sensitive strains. The results indicate that several temperature-sensitive and morphological mutations are recessive and nonidentical.

Genes, Recessive↗

Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics and antimicrobial susceptibilities.

Levels of DNA relatedness were determined by performing DNA-DNA hybridization experiments (S1 nuclease procedure) with 13 human isolates exhibiting various antimicrobial susceptibility patterns which had been identified as Corynebacterium jeikeium by classical tests and the API Coryne system and with reference strains of C. jeikeium and related taxa. Twelve of 13 isolates which formed three genomic groups showed between 22 and 75% relatedness with the type strain of C. jeikeium. One of these genomic groups included all the strains resistant to penicillin and gentamicin and is genomically related to the C. jeikeium type strain at the species level. In addition, the reference strain of "Corynebacterium genitalium" biotype II was found to belong to this genospecies and therefore can be considered as a synonym of C. jeikeium. In contrast, one isolate and the reference strains of "Corynebacterium pseudogenitalium" biotypes C-3 and C-4 which were assigned to C. jeikeium by the API Coryne system were less than 10% related to the C. jeikeium type strain. These nongenomically related strains can be differentiated from the jeikeium-related strains on the basis of positive acidification from fructose and growth under anaerobic conditions. Furthermore, these strains exhibited full susceptibility to penicillin whereas the strains related to the C. jeikeium type strain are resistant to or only moderately susceptible to penicillin. No genomic relationship was found between C. jeikeium-related strains and other lipophilic coryneforms, identified as Corynebacterium accolens or Corynebacterium group G or F. Our study demonstrates the necessity to perform the fructose fermentation test or respiratory-type test for the correct identification of lipophilic coryneforms as C. jeikeium. Although these strains show genomic diversity at the species level, in a practical aspect, biochemical properties as well as antimicrobial susceptibility may allow the classification of such isolates in this single taxon.

Bacterial Typing Techniques↗

Degradation of Escherichia coli chromosome after infection by bacteriophage T4: role of bacteriophage gene D2a.

Mutations in the D2a gene of bacteriophage T4 have recently been shown to result in the stabilization of cytosine-containing phage deoxyribonucleic acid (DNA) made after infection by phage gene 56 (deoxycytidine triphosphatase) mutants. In the experiments reported here, we investigate the role of the D2a gene in the degradation of the host chromosome. We find that if T4 endonuclease II, a product of the phage gene denA, is active, host chromosome degradation appears normal, regardless of the presence of the D2a gene product. However, if T4 endonuclease II is absent, a small amount of host chromosome degradation occurs, but only if the D2a product is present. These results are interpreted in terms of the hypothesis that D2a controls a nuclease which degrades cytosine-containing DNA. Neither D2a nor denA mutations affect the shut-off of host DNA synthesis.

Chromosomes, Bacterial↗

Properties of the deoxyribonucleic acid contained in the defective particle coliphage 15.

Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm(2)) or to mitomycin C (5 mug/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm(3) followed by components with densities of 1.5 and 1.49 g/cm(3). The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 +/- 1% and 46.6 +/- 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 x 10(6) daltons.

Arginine↗

Control of gene function in baceriophage T4. II. Synthes of messenger ribonucleiccid and proei after interrupting deoxyribonucleic acid replication and glucosylation.

Replication of T4 deoxyribonucleic acid (DNA) is known to be required for the onset of transcription of late T4 genes. Once late gene transcription has been initiated, DNA replication is no longer required for maintaining synthesis of late or early T4 messenger ribonucleic acid (mRNA). Late phage proteins (lysozyme and tail fibers) continue to be produced at constant rates after interrupting T4 DNA synthesis. The ability of the host cell to glucosylate the T4 progeny DNA has no demonstrable influence on the rates at which T4 mRNA and late proteins are synthesized after the interruption of DNA synthesis. To explain the requirement of T4 DNA replication for the onset of late gene transcription, we suggest that T4 DNA in a nascent state is mandatory for the initial late gene transcription, or perhaps for late gene transcription throughout the lytic cycle. T4 DNA in a nascent state could be segregated from the bulk of the replicating DNA, used only as template for RNA synthesis, and prevented from being modified by methylation, glucosylation, or maturation processes. The fact that no, or very little, nonglucosylated T4 DNA is extractable from T4LB3-infected CR63 after arresting DNA synthesis does not rule out this possibility.

Centrifugation, Density Gradient↗

Deoxyribonucleic acid polymerase activity associated with strain MC29 tumor virus.

Chick embryo cells infected with strain MC29 tumor virus yielded progeny virus that contained detectable deoxyribonucleic acid (DNA) polymerase within the first 48 hr after infection. The noninfected culture fluids displayed no such enzyme activity when examined in an identical manner. Enzyme activity was greatly stimulated by adding DNA template to the reaction mixture and required all four deoxyribonucleoside triphosphates for full activity. When calf thymus DNA was used to direct synthesis, the DNA polymerase from the MC29 virus catalyzed the formation of DNA product having a higher buoyant density in CsCl. DNA product formed in the reaction directed by Micrococcus lysodeikticus DNA had the same buoyant density as the template DNA.

Animals↗

Polar mutations in the left arm of bacteriophage lambda i434.

By studying complementation between frameshift and nonsense mutants located in the structural genes for the head of bacteriophage lambdai434, we found mutations in gene B which are polar on genes C and D and one mutation in gene E which is polar on gene F.

Acridines↗

Superinfection in bacteriophage S13 and determination of the number of bacteriophage particles which can function in an infected cell.

Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage phiX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.

Binding Sites↗

Gene regulation in N mutants of bacteriophage lambda.

Mutants (N(-)nin) of bacteriophage lambda in which the N gene product is not required for growth on wild-type Escherichia coli do not plate on recA bacterial mutants. Secondary mutants, selected for growth on recA, lie within the immunity region to the right of gene cI and appear identical to the cro mutants of Eisen et al. In an N(+) phage, a cro mutation causes enhanced and prolonged production of lambda exonuclease. N(-)cro phages make no detectable exonuclease, but show an increased rate of specific excision from lysogens and are excluded by P2 prophage. These properties, together with the ability to plate on recA, suggest that N(-)cro phages express genes to the left of N at a rate that is very low but higher than that for N(-)cro(+) phages. N(-)nin phages can integrate at the normal site on the bacterial chromosome, but specific excision from lysogens is immeasurably low.

Binding Sites↗