Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.     

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.     

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema     

Fatty acid composition of the lipids of streptomycetes and their nocardia-like mutants

The models "population--Nocardia-like spontaneous mutants" for 14 cultures of Streptomycetes and Streptoverticillium were used to assess the fatty acid composition of lipids as a criterion for generic differentiation of Streptomyces and Nocardia cultures. The composition of fatty acids in the Nocardia-like mutant Str. kanamyceticus RIA-771 was found to be identical with that of Nocardia asteroides RIA-43 and Nocardia brasiliensis RIA-440, i. e. generic chemotaxonomic traits "overlapped" in the process of spontaneous intraspecial variability. In different strains of one and the same species as well as in five different species, the quantitative composition of fatty acids either hardly changed in the process of intraspecial variability or the ratio between fatty acids changed, so that Nocardia-like mutants resembled typical cultures of the Nocardia genus in this characteristics. The results suggest that the quantitative composition of fatty acids of lipids cannot be regarded as a sufficiently reliable criterion for generic differentiation of Steptomyces and Nocardia cultures. This trait should be used only for additional characterization of cultures.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

Procedure for the isolation of mutants of Bacillus subtilis with defective cytoplasmic membranes

A procedure has been devised to isolate mutants of Bacillus subtilis with structurally defective membranes. The procedure used to screen for the mutants involved comparison of the stability of protoplasts of the mutant with those of the wild type in a medium of sufficient osmotic strength to stabilize wild-type protoplasts. Mutagenized cells were grown as clones on agar plates, and then replicated onto plates containing 0.5 m lactose, which is sufficient to stabilize wild-type protoplasts. The colonies on the lactose-containing plates were then treated with lysozyme to convert the cells to protoplasts. Colonies of wild-type protoplasts remained opaque; however, colonies of mutant protoplasts lysed and became clear. Twenty-nine osmotically fragile mutants were isolated in this manner; the membranes of several mutants were found to contain alterations in the composition of their proteins or lipids.     

Enzymatic studies of high stearic acid sunflower seed mutants

Three high stearic acid sunflower (Helianthus annuus L.) mutants, CAS-3, CAS-4 and CAS-8, accumulating 28, 15 and 14 % of stearic acid in the seed lipids have been biochemically characterised. In vivo conversion rate of palmitic acid into stearic acid is not altered in the mutants but the conversion rate of stearic acid into oleic acid shows a reduction that correlated with the total stearic acid content of seed lipid mutants. Two enzymatic activities are found to be involved in the mutant phenotype, the acyl-ACP thioesterase (EC 3.1.2.14) and the stearoyl-ACP desaturase (EC 1.12.99.6). Our data suggest that the high stearic phenotype is due to the combined effect of a reduced stearoyl-ACP desaturase activity and an acyl-ACP thioesterase with higher activity on stearoyl-ACP. The same thioesterase activity increment, found on stearoyl-ACP, was also found on palmitoyl-ACP, suggesting that the affected thioesterase activity could be a FatB type.     

Isolation, characterization, and mapping of Bacillus subtilis 168 germination mutants.

After mutagenesis with nitrosoguanidine, germination mutants of Bacillus subtilis 168 were selected by killing, with heat, spores that germinated at 42 C and collecting survivors at 30 C. The germination properties of nine mutants variously affected in amino acid biosynthesis and sugar utilization were studied in detail. They were divided into two groups: (i) Ger-ALA mutants, failed to germinate in 10 mM L-alanine but germinated in complex media (some of these mutants were temperature sensitive); (ii) Ger-PAB mutants, germinated poorly, even in complex media, suggesting that they were blocked in important germination functions. All the mutants failed to germinate in L-alpha-amino-n-butyrate or L-valine (including temperature-sensitive mutants only at the restrictive temperature) showing that there is a step necessary for germination affected by all three acids. The mutants had normal growth rates, indicating that the defective gene products were specific for germination functions. These defects were not identified. Eight of the mutants were mapped by transduction with phage PBS-1. The recombinants were scored either by observations, by microscopy of phase darkening of the spores, or by a plate test involving the reduction of tetrazolium by heated colonies of spores. Five of the mutations, of at least three phenotypes, were between thr-5 and cysB3 away from all the sporulation markers that have been previously mapped. A linked ald (alanine dehydrogenase) locus was on the other side of thr-5. The other Ger markers were located in at least two additional positions. Auxotrophic strains that were used for mapping germinated normally, but germination of the Ger mutants differed slightly in different genetic backgrounds.     

Drug resistance and membrane alteration in mutants of mammalian cells.

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.     

Relation entre structure, composition et fractionnement par le Triton X-100 de la lamelle chloroplastique de la souche sauvage et de deux mutants non photosynthetiques de Chlamydomonas reinhardti

Relationship of structure, composition and Triton X-100 fractionation of chloroplas lamellae in wild type and two non-photosynthetic mutant strains of Chlamydomonas reinhardti

In order to provide information on the link between the two photosystems studies on the mode of action of Triton X-100 has been carried out on mutants, strains ac 21, Fl 15 and wild type of Chlamydomonas reinhardti. Experiments show that the release of Photosystem I particles from mutant chloroplast fragments needs less Triton X-100 than wild type does and that, compared to wild type, the chloroplast fragments of mutants appear to be deficient in carotenoids (ac 21) or in lipids (Fl 15). It is possible, therefore, to correlate the easier splitting of the mutant membrane by detergent with a decrease in the amount of these compounds (carotenoids and lipids) in mutant strains.

The following interpretation is proposed: (a) some of the carotenoids could be part of the hydrophobic sites on Photosystem I subchloroplast particles; (b) some polar lipids could be linked to these sites; (c) Triton X-100 could, in a competitive way, replace the membrane lipids linked to the hydrophobic sites of subchloroplast particles. It seems probable that anomalies in the mutant behaviour in regard to the Triton X-100 action are related to membrane structural defects in these mutants.     

Lipid interaction of diphtheria toxin and mutants. A study with phospholipid and protein monolayers

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm). DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm). The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.     

Uncoupler-resistant mutants of bacteria.

The chemiosmotic model of energy transduction offers a satisfying and widely confirmed understanding of the action of uncouplers on such processes as oxidative phosphorylation; the uncoupler, by facilitating the transmembrane movement of protons or other compensatory ions, reduces the electrochemical proton gradient that is posited as the energy intermediate for many kinds of bioenergetic work. In connection with this formulation, uncoupler-resistant mutants of bacteria that neither exclude nor inactivate these agents represent a bioenergetic puzzle. Uncoupler-resistant mutants of aerobic Bacillus species are, in fact, membrane lipid mutants with bioenergetic properties that are indeed challenging in connection with the chemiosmotic model. By contrast, uncoupler-resistant mutants of Escherichia coli probably exclude uncouplers, sometimes only under rather specific conditions. Related phenomena in eucaryotic and procaryotic systems, as well as various observations on uncouplers, decouplers, and certain other membrane-active agents, are also briefly considered.     

Chemotaxis mutants of Spirochaeta aurantia.

Five Spirochaeta aurantia chemotaxis mutants were isolated. One mutant (the che-101 mutant) never reversed, one (the che-200 mutant) flexed predominantly, two (the che-300 and che-400-1 mutants) exhibited elevated reversal frequencies, and one (the che-400 mutant) exhibited chemotactically unstimulated behavior similar to that of the wild-type strain. The che-101 and che-400 mutants were essentially nonchemotactic, whereas the che-200, che-300, and che-400-1 mutants showed impaired chemotactic responses. Protein methylation in response to attractant addition appeared normal in all of the mutants. Compared with the wild type, all of the mutants exhibited significantly altered membrane potential responses to the attractant xylose.     

Ultrastructure,composition of neutral lipids and their fatty acids of Candida tropicalis strain D-2 mutants resistant to the polyene antibiotic nystatin

This report deals with data on the cell ultrastructure of Candida tropicals strain D-2 mutants resistant to the polyene antibiotic, nystatin, and with an analysis of the fractional composition of neutral lipids and their fatty acids. The ultrastructural organization of the mutant cells is characterized by thickening of the cell wall and formation of invaginations into the cytoplasm, the appearance of new formations, large vacuoles, and reduction of the system of mitochondrial cristae. Lipids of nys^r mutants differ from those of the nys^s variant in having a decreased content of steroids and some fractions of neutral lipids. Certain nys^r mutants manifest difference in the relative amounts of saturated and unsaturated fatty acids (C_16:0, C_16:1, C_18:0, C_18:1).     

Pleiotropic, extragenic suppression of dna mutants in Bacillus subtilis.

Thermosensitive (dna) mutants of Bacillus subtilis defective in deoxyribonucleic acid replication can be divided into two groups on the basis of their ability to spontaneously yield secondary mutants with an HDS phenotype (thermoin-sensitivity and resistance to aryl-azo-pyrimidines) at frequencies higher than 10(-8). Such a phenotype is due to alleles at the hds locus (mapping close to cysA), which act as extragenic pleiotropic suppressors. HDS suppressibility has been used as a screening tool to identify new dna strains among uncharacterized temperature-sensitive mutants.     

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.