Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance

Summary Escherichia coli cells carrying the dnaK756 mutation, were inactivated at 52°C faster than control cells. This suggests that the intact dnaK gene product plays a role in protecting the cell from lethal damage at 52°C. The effect of the dnaK mutation on induced thermotolerance was examined. Prior heat shock at 42°C greatly lowered the subsequent inactivation rate in both mutant and control cells. This result suggests that, although produced in large amounts in response to thermal stress, mutation in the DnaK protein has little or no effect on induced thermotolerance.     

Induction and decay of thermosensitivity in the flesh fly,Sarcophaga crassipalpis

When pharate adults of the flesh fly Sarcophaga crassipalpis are exposed to 40°C for 4 h they become more tolerant of high temperatures that are normally lethal (thermotolerance). In contrast, a 1-h exposure to 45°C decreases tolerance to a subsequent high temperature challenge (thermosensitivity). While control flies experience little mortality when held at 35°C for 24–48 h the thermosensitized flies die when exposed to 35°C. Sensitivity to a second thermal challenge slowly decays over a 72-h period. The acquisition of thermotolerance prevents the development of thermosensitivity. Brains from thermosensitized flies cultured at 43°C express the 72-kDa heat-shock protein and normal protein synthesis is inhibited. This implies that development of thermosensitivity is not associated with a loss in the capacity to express the 72-kDa heat-shock protein.Abbreviations ICN ICN Biomedicals, Inc. PO Box 19536, Irvine, CA 92713-9921 - LD light dark cycle - LT₅₀ time required to kill 50% of the test animals - SDS sodium dodecyl sulfate - TRIS Tris(hydroxymethyl)aminomethane     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

Conditionallethality of Escherichia coli strains carrying dnaE anddnaQ mutations

Summary A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at28° C but not at 30° C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. a dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperaturesensitive graowth mutations were conbined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Life-history traits of the acarophagous lady beetle,<Emphasis Type="Italic">Stethorus japonicus</Emphasis> at three constant temperatures

Stethorus japonicusKamiya (Coleoptera: Coccinellidae) is an indigenous ladybird beetle in Japan, which feeds on many spider mite species. We evaluated the development, survivorship and life-history parameters of this lady beetle on a diet of eggs of the two-spotted spider mite, Tetranychus urticae Koch (red form) (Acari: Tetranychidae). In addition, the effect of short photoperiod on its reproduction was assessed. Survival rates from egg to adult were more than 71% at temperatures between 17.5 and 30 °C. The highest immature mortality was 100% at 35 °C followed by 76% at 15 °C and 52% at 32.5 °C. The lower threshold temperature for development from egg to egg-laying adult was 13.0 °C and the thermal constant was calculated as 238.7° days. Based on these data, the maximum number of generations that could complete development in a year under field conditions in Ibaraki, central Japan, would be between five and seven. The intrinsic rates of natural increase (r_m) were 0.093 at 20 °C, 0.156 at 25 °C and 0.241 at 30 °C. Reproductive diapause was induced at photoperiods with light phases shorter than 13 h at 18 °C.     

Desulfurella acetivorans gen. nov. and sp. nov. —a new thermophilic sulfur-reducing eubacterium

A new type of thermophilic cyanobacterial mat, rich in elemental sulfur and containing large numbers of sulfur-reducing bacteria able to utilize different growth substrates at 55° C, was found in the Uzon caldere (Kamchatka). One of the largest groups among these organisms were acetate-oxidizing sulfur-reducing bacteria, numbering 10⁶ cells · cm^–3 of mat. The pure culture of a sulfur-reducing eubacterium growing on acetate was isolated. Cells of the new isolate are Gram-negative short rods, often in pairs, motile, with a single polar flagellum. The optimal temperature for growth is 52 to 57° C, with no growth observed at 42 or 73° C. The pH optimum is 6.8 to 7.0. The new isolate is demonstrated to be a true dissimilatory sulfur reducer: it is an obligate anaerobe, it is unable to ferment organic substrates and it can use no electron acceptors other than elemental sulfur. Acetate is the only energy and carbon source, and H₂S and CO₂ are growth products. No cytochromes were detected. The G+C content of DNA is rather low, only 31.4 mol%. Thus, morphological and physiological features of the new isolate are quite close to those of Desulfuromonas. But on the grounds of a significant difference in the G+C content of DNA, the absence of cytochromes and because of its thermophilic nature, a new genus Desulfurella is proposed with the type species Desulfurella acetivorans.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Cyclic CO(2) emissions during the high temperature pulse of fluctuating thermal regime in eye-pigmented pupae of Megachile rotundata

Megachile rotundata (Hymenoptera: Megachilidae), the primary pollinator used in alfalfa seed production, may need to be exposed to low-temperature storage to slow the insects' development to better match spring emergence with the alfalfa bloom. It has been demonstrated that using a fluctuating thermal regime (FTR) improves the tolerance of pupae to low temperatures. Carbon dioxide emission rates were compared between four different FTRs, all with a base temperature of 6 °C and a daily high-temperature pulse. Four different high-temperature pulses were examined, 15 or 25 °C for 2 h and 20 °C for 1 or 2 h. A subset of pupae at the FTR base temperature of 6 °C exhibited continuous gas exchange and, once ramped to 20 or 25 °C, shifted to cyclic gas exchange. As temperatures were ramped down from the high-temperature pulse to 6 °C, the pupae reverted to continuous gas exchange. The following conclusions about the effect of FTR on the CO₂ emissions of M. rotundata pupae exposed to low-temperature storage during the spring incubation were reached: 1) the high temperature component of the FTR was the best predictor of respiratory pattern; 2) neither pupal body mass nor days in FTR significantly affected which respiratory pattern was expressed during FTRs; 3) cyclic gas exchange was induced only in pupae exposed to temperatures greater than 15 °C during the FTR high temperature pulse; and 4) a two hour pulse at 25 °C doubled the number of CO₂ peaks observed during the FTR pulse as compared to a two hour pulse at 20 °C.     

Evaluation of different temperature-sensitive mutant phenotypes ofSalmonella enteritidis as vaccine potentials

Temperature-sensitive (ts) mutants ofSalmonella enteritidis were isolated after mutagenesis with UV light and enrichment with antibiotic. Mutants were characterized according to their growth profile at the permissive (28°C) and the nonpermissive (37°C) temperatures, persistence of surface antigens, reversion frequencies, and potentials for inducing humoral immunity and protection against challenge with the parental wild-type (wt) in mice. We obtained 32 strains ofS. enteritidis able to grow well at 28°C, but capable of only limited or no replication at 37°C. The ts mutants were positive for factor 9 in an agglutination assay and were susceptible to infection with phage P22. Three mutants of different phenotypes were selected for protection studies. A single intraperitoneal (i.p.) immunization with any of the mutants studied induced significant protection from i.p. challenge with 100 LD₅₀ of the wt strain.     

Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures

The effect of temperature and O₂ saturation on the production of recombinant proteins -galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O₂ consumption and product expression were measured at temperatures between 22° C and 35° C. The results indicated that possible O₂ limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27° C to 25° C. The expression level of the recombinant proteins at 27° C was similar to that obtained at 22° C and 25° C; lower protein yields were obtained at 30° C. An increase in temperature from 22° C to 27° C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells. Correspondence to: J. Shiloach     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

Life history study of Thrips setosus

Development, reproduction and population growth of Thrips setosus Moulton (Thysanoptera, Thripidae), reared on a leaf of kidney bean, was studied under six different constant temperatures, and the effect on reproduction of short photoperiod during immature stages was examined. Survival rates from hatch to adult were more than 67.5% at temperatures between 17.5 and 27.5 °C, but less than 55% at 30 °C. Developmental rates increased linearly as rearing temperature increased. A total of 181.1 degree-days, above a developmental zero of 12.5 °C, were required to complete development from egg to adult oviposition. These data were related to records of field temperatures in Kurashiki in western Japan, and an estimate produced that, under outdoor conditions, a maximum of between seven and 12 generations could have developed annually between 1990 and 1999. There were no significant differences in mean adult longevity and mean fecundity among three temperatures (20, 22.5 and 25 °C). The intrinsic rate of natural increase (r _m) was 0.1997 at 25 °C. Reproductive diapause was induced by a photoperiod less than 12 h at 20 °C.     

Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52°C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO₂), converted DBTO₂ to 2-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30–52°C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50°C and DBT desulfurization (2-HBP production) at 40°C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.     

Isolation and Primary Identification of Methylotrophic Yeast Hansenula polymorpha Mutants for Peroxisome Biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorphaHF246leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45°C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45°C but could grow at optimal temperature (37°C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10pex10 mutants (4ts mutants among them); group 2 included 19 mutants that failed to complement otherpex testers: 1 pex1; 2 pex4(1ts); 6 pex5(5ts); 3 pex8; 1 pex13; 6 (3ts) pex19; group 3 contained 22 multiple mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30°C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. Analysis of the spore population from the hybrids of remaining 14 mutants with the pex tester demonstrated the presence of methanol-utilizing segregants, which indicates mutation localization in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed us to localize this mutation in the only PEX gene (PEX1 or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Pollen selection for low temperature adaptation in tomato

Summary Pollen selection experiments were conducted in tomato to determine the effects of low temperature conditions during pollination on the rate of root elongation of the progeny. Pollen was harvested from an F₁ interspecific hybrid between a high altitude Lycopersicon hirsutum accession and the cultivated tomato L. esculentum. The pollen was applied to stigmas of malesterile L. esculentum plants maintained in growth chambers set at either 12°C/7°C or 24°C/18°C. BC₁ seeds from the low and normal temperature crosses were germinated and root elongation rate was measured at either 9°C or 24°C. At 9°C, the rate of root elongation for progeny of the low temperature crosses was higher than for progeny of crosses at normal temperatures; at 24°C the rate of root elongation was similar for the two crossing treatments. To compare the temperature responses of the two backcross populations we also calculated the relative inhibitory effect of low temperature on the rate of root elongation: the ratio between the rate of root elongation at 9°C to that at 24°C. Root elongation of seedlings from the low temperature crosses was less inhibited by the cold than root elongation for progeny of the normal temperature crosses. These results suggest a relationship between pollen selection at low temperatures and the expression of a sporophytic trait under the same environmental stress.     

Prolonged environmental stress via a two step process selects mutants of Escherichia,Salmonella and Pseudomonas that grow at 54°C

A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48°C with nalidixic acid selected mutants (T48) able to grow at 48°C. A prolonged incubation at 54°C of the T48 mutants selected mutants (T54) able to grow at 54°C. These mutants were susceptible to the same bacteriophages as the original mesophilic strains. Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48°C or 54°C or on a minimal agar with glucose at 37°C. The T48 alleles mapped in the gyrA region of E. coli or S. typhimurium chromosome. In S. typhimurium the T54 alleles, which permit growth at 54°C, were shown by cotransductional analysis to be linked to gyrA.     

Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

Negative complementation among ad2 mutants of yeast

Summary Ad ₂ remedial mutants of Saccharomyces cerevisiae which showed conditional growth at 35°C or with 1 M KCl at 25° C or 35° C, were crossed with other remedial and non-remedial mutants in all pairwise combinations. 19 remedials (11 K ₂₅, 5 temp. and 3 K ₃₅) and 189 non-remedial mutants were used. The standard conditions used were incubation at 25° C in the absence of adenine.Among 1625 combinations there were 367 (22.6%) cases of negative complementation. In these cases the diploids were unable to grow under the same conditions which permitted a strong growth of the remedial haploid.No negative complementation was observed among K ₂₅×K ₂₅ combinations. When only one haploid partner was remedial the temp. ¢ non-remedial combinations showed a rate 2.5 times higher than that observed among K ₂₅ × non-remedial combinations. When the remedial haploid was a K ₃₅ the incidence was the same as that noted with temp. × non-remedial combinations. The high incidence observed in the latter temp. combinations was not subject to influence attributable to strain; the incidence being the same whether the remedial partner belonged to the a or to the mutant strains. The K ₂₅ × non-remedial combinations, on the other hand, showed such an influence.For an explanation of the mechanism involved in negative complementation it was postulated that, assuming non-random monomer aggregation, some mutant pairs would form only active heterologous multimers while others, due to the nature of their mutation, would form only inactive aggregates which entrap all the remedial monomers. It is thought that this postulate may have some application in explaning the complementation mechanism in general.Mit Unterstützung der Deutschen Forschungsgemeinschaft.