Temperature-sensitive mutants of Neisseria gonorrhoeae.

Nine temperature-sensitive (Ts) mutants of Neisseria gonorrhoeae strain 49191 were isolated. They were proven to be different from each other by results of transformation experiments. None of the Ts mutations appeared to be linked to antibiotic resistance genes from strain 24392. However, Ts-9 demonstrated 8% linkage with a nalidixic acid resistance marker from strain RW-2.     

Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine-cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 x 10(5) cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 greater than 10(7) cells), even though it grew well in vivo and in mouse serum at 37 degrees C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.     

Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.     

Correlation between polyploidy and auxotrophic segregation in the imperfect yeast Candida albicans.

In order to clarify the relationship between polyploidization and the capability of phenotypic switching in the imperfect yeast Candida albicans, two types of variants were isolated as segregants from a fusant, which produced a proportion of the cell population with a higher ploidy than the rest, either in a temperature-dependent or -independent manner, when incubated at low (28 degrees C) and high (37 degrees C) temperatures. In the case of the temperature-dependent type of variants, high-ploidy cells appeared at 37 degrees C but rarely at 28 degrees C. This phenotype was named Pldts (temperature-sensitive polyploidization), and the temperature-independent phenotype was called Pld-. The appearance of high-ploidy cells in the culture of the Pldts strain at 37 degrees C was accompanied by a significant increase in the frequency of auxotrophic variants; these variants probably occur as a result of segregation of auxotrophic markers from the heterozygous to the homozygous state. Both Pldts and Pld- phenotypes were recessive in a fusion with a Pld+ parent. An adenine auxotrophic marker (ade1) was introduced into a Pldts strain in a heterozygous state, and the individual high-ploidy cells of this strain, grown at 37 degrees C, were micromanipulated to form colonies, which consisted of red and white sectors appearing at high frequency on a pink background. When the ade1 auxotrophy was introduced into Pld- strains, frequently sectored colonies were produced. These results suggested an increased level of chromosome missegregation in both types of Pld mutants. Analyses by pulsed-field gel electrophoresis of Ade-segregants, derived from a micromanipulated high-ploidy cell of a Pld(ts) strain, suggested the occurrence of nonreciprocal recombination, some of which includes chromosome loss.     

Auxotrophic mutations related to symbiotic properties of Rhizobium meliloti strain L5-30.

Mutants isolated from effective R. meliloti strain L5-30 which required histidine (his-240), arginine+uracil (arg-55) and cysteine (cys-243, cys-244 and cys-246) showed also loss of effectiveness. Mutant requiring isoleucine+valine (ilv-74) was non-infective. Relation of the metabolic deficiency to the symbiotic properties of these mutants was tested comparing symbiotic response of their prototrophic revertants and transductants. It was found that all revertants and transductants of the strain his-240 were effective which suggests that histidine deficiency was the cause of their ineffectiveness. All revertants and transductants of the cysteine mutants were still ineffective. This result indicates two independent mutations which were not cotransductible. Prototrophic revertants of the mutant arg-55 were ineffective whereas 56.9 percent of transductants appeared effective suggesting close linkage of two mutations. i.e. auxotrophic and the other concerned with symbiotic effectiveness. Though one of 69 prototrophic transductants obtained from the non-nodulating mutant ilv-74 remained non-nodulating, it seems that changes in nodulating ability of the mutant are related to the auxotrophic requirements.     

Chromosomal map of Pseudomonas putida PPN, and a comparison of gene order with the Pseudomonas aeruginosa PAO chromosomal map

The generalized transducing phage Pf16h2 has been used to confirm linkage relationships of chromosomal markers of Pseudomonas putida previously determined from their time-of-entry in Hfr crosses, and to map new auxotrophic mutations. By means of spot matings using Hfr donors of known origin of transfer, catabolic markers forming part of a closely linked group of operons referred to as a superoperonic cluster have been shown to be chromosomally located and their map positions determined. R-prime-mediated interspecific complementation has been used to equate functionally 21 auxotrophic loci in P. putida and P. aeruginosa, and the distribution of these loci on the two genetic maps has been compared. While both maps reveal that auxotrophic markers are largely restricted to about 40% of the chromosome and that auxotrophic markers of similar phenotype are not clustered, there is evidence of at least seven chromosomal rearrangements since divergence from a presumed common ancestor.     

Isolation and characterization of temperature-sensitive mutants of Sendai virus

Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.     

Expanded Linkage Map of VIBRIO CHOLERAE

An expanded linkage map of the Vibrio cholerae classical strain 162 chromosome has been prepared using a variety of new auxotrophic mutants. The chromosome consists of a single, linear linkage group. The map consists of 17 markers, which have been ordered; 20 mutational sites, which are tentatively ordered; five markers (ura-1, ser-2, mal-1, man-1, suc-1), which are linked but unordered; and three mutations (aro-2, cys-2 and cys-6) which showed little or no linkage. A proposal is made to standardize genetic nomenclature in V. cholerae genetic studies.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations. Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Extension of a chromosome linkage group of Proteus mirabilis.

Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and R1drd19. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 degrees C before washing and plating on selective media. Final incubation was also at 30 degrees C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines.

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Temperature-sensitive beta-lactam-tolerant mutants of Escherichia coli

Seven temperature-sensitive penicillin-tolerant mutants of Escherichia coli strain LD5 (thi lysA dapD) were isolated and characterized. Treatment with beta-lactams caused lysis of the mutants at 30 degrees C. Although growth of the mutants at 42 degrees C was inhibited by beta-lactams, no lysis occurred. The mutants were also slightly tolerant to D-cycloserine at 42 degrees C but lysed readily when deprived of diaminopimelate or when treated with moenomycin. The minimum inhibitory concentrations of various antibiotics were the same for the mutants and their parent. The mutations conferring penicillin tolerance were phenotypically suppressed in the presence of a variety of compounds which may act as chaotropic or antichaotropic agents. No defects in penicillin-binding proteins and peptidoglycan hydrolases were detected. Temperature-resistant revertants of the mutants were no longer tolerant to penicillin-induced autolysis at 42 degrees C. The mutations in five isolates were localized to the 56 to 61 min region of the E. coli linkage map and to the 44 to 51 min region in the case of two other isolates.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.