Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C. At the restrictive temperature, ypt1ts mutants accumulated ER membranes, small vesicles, and unprocessed invertase, and they exhibited cytoskeletal defects and an enhanced 45Ca2+ uptake. Similar alterations were seen in YPT1-depleted cells. The ypt1ts mutant cells could be rescued from growth arrest by increasing extracellular Ca2+, and, even at the permissive temperature, they displayed increased trifluoperazine sensitivity.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii.

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to aw 0.93 were more rapid than in unsupplemented media (aw 0.99). Although growth in unsupplemented medium was lower at 35 degrees C, incubation at 21 degrees C or 35 degrees C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 micrograms potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35 degrees C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35 degrees C than in cultures incubated at 21 degrees C in media both with and without 300 micrograms potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at -18 degrees C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 micrograms potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21 degrees C. Sensitivity to freezing increased when cultures were incubated at 21 degrees C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Mutants of Bacillus subtilis affected in spore outgrowth.

Six mutants of Bacillus subtilis 168 that are temperature-sensitive in spore outgrowth were isolated. The outgrowth process proceeds normally at 35 degrees C, but at the non-permissive temperature (47 degrees C) it is arrested at a specific stage characteristic for each mutant strain. The mutants are not altered in vegetative growth whether at 35 degrees C or at 47 degrees C. They were characterized for their ability to synthesize RNA, proteins and DNA during outgrowth. A mutant defective in spore germination was also isolated; less than 5% of its spores can germinate at any of the temperatures tested. The mutations were mapped by means of transduction and transformation. The isolation of a number of outgrowth mutants which map at different loci and which affect outgrowth at different times is discussed in relation to the regulation of this process.     

Genetic characterization of genes encoding enzymes catalyzing addition of phospho-ethanolamine to the glycosylphosphatidylinositol anchor in Saccharomyces cerevisiae

MPC1/GPI13/YLL031C, one of the genes involved in the addition of phospho-ethanolamine to the glycosylphosphatidylinositol (GPI) anchor core, is an essential gene. Three available temperature-sensitive mutant alleles, mpc1-3, mpc1-4, and mpc1-5, displayed different phenotypes to each other and, correspondingly, these mutants were found to have different mutations in the MPC1 ORF. Temperature-sensitivity of mpc1-5 mutants was suppressed by 5 mM ZnSO(4) and by 5 mM MnCl(2). Multicopy suppressors were isolated from mpc1-5 mutant. Suppressors commonly effective to mpc1-4 and mpc1-5 mutations are PSD1, encoding phosphatidylserine decarboxylase, and ECM33, which were found to suppress the temperature-sensitive phenotype shown by the fsr2-1 and las21delta mutants, those of which have defects in the GPI anchor synthesis. PSD2, encoding another phosphatidylserine decarboxylase that is localized in Golgi/vacuole, was found to be able to serve as a multicopy suppressor of mpc1 and fsr2-1 mutants but not of the las21 delta mutant. In contrast to psd1delta, psd2delta showed a synthetic growth defect with mpc1 mutants but not with fsr2-1 or las21delta. Furthermore, psd1delta psd2delta mpc1 triple mutants did not form colonies on nutrient medium unless ethanolamine was supplied to the medium, whereas psd1delta psd2 delta fsr2-1 or psd1delta psd2 delta las21delta triple mutants grew on nutrient medium without supplementation of ethanolamine. These observations suggest that Mpc1 preferentially utilizes phosphatidylethanolamine produced by Psd2 that is localized in Golgi/vacuole. fsr2-1 dpl1 Delta psd1delta strains showed slower growth than fsr2-1 dpl1delta psd2 delta, suggesting that Fsr2 enzyme depends more on Dpl1 and Psd1 for production of phosphatidylethanolamine. Las21 did not show preference for the metabolic pathway to produce phosphatidylethanolamine.     

Morphology and patterns of protein synthesis during sporulation of Bacillus subtilis Eryr spo(Ts) mutants.

Erythromycin-resistant (Eryr) mutants of Bacillus subtilis 168 fail to sporulate at high temperature (47 degrees C) but sporulate normally at 30 to 35 degrees C. They also fail to sporulate at any temperature in the presence of 2.5 micrograms of erythromycin per ml. Neither of these nonpermissive conditions appears to affect vegetative growth, and the periods of sensitivity to both conditions extend from 40 to 90% of the sporulation period. At 47 degrees C, net incorporation of methionine and phenylalanine in postexponential Eryr and 168 cells was similar, and fractionation of the labeled products by polyacrylamide gel electrophoresis gave patterns in which many of the bands produced by mutant and parental cells coincided. However, distinct differences were seen, and since no spore-specific morphogenesis occurred in the Eryr cells at 47 degrees C, a selective defect in spore gene expression was inferred. At 35 degrees C plus erythromycin, spore morphogenesis proceeded normally until forespores were produced and then ceased, coincident with a marked increase in sensitivity of total protein synthesis to erythromycin. The effects seem to be nonspecific, therefore, and may indicate a change in cell permeability or ribosomal sensitivity to erythromycin.     

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 degrees C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 degrees C, a transient increase in both cytochrome c oxidase and DNA polymerase gamma was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular cross-section as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 micrograms/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 degrees C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 degrees C or during treatment with 1 mM butyrate, suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.     

Interaction of actinogelin with actin. No nucleation but high gelation activity

Nucleation activity of actin polymerization of actinogelin, a calcium-sensitive F-actin cross-linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl-actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization. By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F-actin through its end(s), and cross-links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F-actin and actinogelin. Under non-gelling high Ca2+ conditions, binding of actinogelin along the side of F-actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination. Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20 degrees C was 6 micrograms/ml for 250 micrograms/ml of actin. Comparing this value with those of the other actin cross-linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; more than 50 micrograms/ml of the protein was required for gelation. At 37 degrees C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20 degrees C, but this was still higher than that of gizzard alpha-actinin at 20 degrees C. Thus, role of actinogelin as an efficient and Ca2+-regulated cross-linker of microfilaments was substantiated.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid composition and membrane function in phosphatidylserine decarboxylase mutants of Escherichia coli.

Temperature-sensitive conditional lethal mutants in phosphatidylserine decarboxylase (psd) accumulate large amounts of phosphatidylserine under nonpermissive conditions (42 degrees C) prior to cell death. In addition, the ratio of cardiolipin to phosphatidylglycerol is increased. At an intermediate temperature (37 degrees C), high levels of phosphatidylserine can be maintained with little effect on cell growth or viability. Under these conditions, both the rate of induction and the function of the lactose transport system are normal. At 42 degrees C addition of Mg2+ or Ca2+ to mutant cultures produces a partial phenotypic suppression. Growth is prolonged and the filaments normally present at 42 degrees C do not form. Upon transfer to the nonpermissive temperature, there is a considerable lag before accumulation of phosphatidylserine begins and the growth rate is affected. Based on the kinetics of heat inactivation of phosphatidylserine decarboxylase activity in extracts, in intact nongrowing cells, and in growing cells, it appears that the enzyme newly synthesized at 42 degrees C is more thermolabile in vivo than enzyme molecules previously inserted into the membrane at the lower temperature. Thus, the older, stable enzymatic activity must be diluted during growth before physiological effects are observed.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Controlled Growth of Chinese Hamster Ovary Cells and High Expression of Antibody-IL-2 Fusion Proteins by Temperature Manipulation

Growth and the expression of the anti-ErbB2 scFv-Fc-IL2 fusion protein in Chinese hamster ovary (CHO) cells were in association at 37 degrees C. The expression of the fusion protein was no more than 25 microg/ml. At 30 degrees C the cell growth was arrested but the cells continued to produce the fusion protein up to 60-80 microg/ml. About 50% of CHO cells were rapidly blocked in G2/M phase after the temperature was shifted from 37 to 30 degrees C. Lowering temperature resulted in cell growth arrest, but maintained cell viability for a longer time and enhanced the production of the antibody-IL-2 fusion protein in CHO cells.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Calcium- and calcineurin-independent roles for calmodulin in Cryptococcus neoformans morphogenesis and high-temperature growth

The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37 degrees C. One insertion mutant, cam1-ts, that displayed a growth defect at 37 degrees C and hypersensitivity to the calcineurin inhibitor FK506 at 25 degrees C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3' untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37 degrees C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Delta diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37 degrees C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37 degrees C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Filamentation promotes F'lac loss in Escherichia coli K12.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.