The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis.

We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102. At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis. These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis.     

Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus ΔltaS mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, ΔltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of ΔltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the ΔltaS mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the ΔltaS mutation was found to be synthetically lethal with the ΔtagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.     

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30°C and shows extensive vegetative petite induction by UV irradiation at 30°C or when cultivated at a higher temperature (37°C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37°C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37°C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.     

ftsE(Ts) Affects Translocation of K+-Pump Proteins into the Cytoplasmic Membrane of Escherichia coli

The ftsE(Ts) mutation of Escherichia coli causes defects in cell division and cell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of KdpA, Kup, and TrkH, all of which proved functional in vivo as K⁺ ion pumps, in the mutant cells. During growth at 41°C, these proteins were progressively lost from the membrane fraction. The reduction in the abundance of these proteins inversely correlated with cell growth, but the preformed proteins in the membrane were stable at 41°C, indicating that the molecules synthesized at the permissive temperature were diluted in a growth-dependent manner at a high temperature. Pulse-chase experiments showed that KdpA-PhoA was synthesized, but the synthesized protein did not translocate into the membrane of the ftsE(Ts) cells at 41°C and degraded very rapidly. The loss of KdpA-PhoA from the membrane fractions of ftsE(Ts) cells was suppressed by a multicopy plasmid carrying the ftsE⁺ gene. While cell growth stopped when the abundance of these proteins decreased 15-fold, the addition of a high concentration of K⁺ ions specifically alleviated the growth defect of ftsE(Ts) cells but not cell division, and the cells elongated more than 100-fold. We conclude that one of the causes of growth cessation in the ftsE(Ts) mutants is a defect in the translocation of K⁺-pump proteins into the cytoplasmic membrane.     

Escherichia coli HU protein has a role in the repair of abasic sites in DNA

HU is one of the most abundant DNA binding proteins in Escherichia coli. We find that it binds strongly to DNA containing an abasic (AP) site or tetrahydrofuran (THF) (apparent K_d ≈50 nM). It also possesses an AP lyase activity that cleaves at a deoxyribose but not at a THF residue. The binding and cleavage of an AP site was observed only with the HUαβ heterodimer. Site-specific mutations at K3 and R61 residues led to a change in substrate binding and cleavage. Both K3A(α)K3A(β) and R61A(α)R61A(β) mutant HU showed significant reduction in binding to DNA containing AP site; however, only R61A(α)R61A(β) mutant protein exhibited significant loss in AP lyase activity. Both K3A(α)K3A(β) and R61K(α)R61K(β) showed slight reduction in AP lyase activities. The function of HU protein as an AP lyase was confirmed by the ability of hupA or hupB mutations to further reduce the viability of an E. coli dut(Ts) xth mutant, which generates lethal AP sites at 37°C; the hupA and hupB derivatives, respectively, had a 6-fold and a 150-fold lower survival at 37°C than did the parental strain. These data suggest, therefore, that HU protein plays a significant role in the repair of AP sites in E. coli.     

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature

Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA^Lys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNA^Lys did not alter the percentages of the free-, charged- or peptidyl-tRNA^Lys species compared with those found in strains that did not overproduce tRNA^Lys. At 41°C, however, despite increases in the level of peptidyl-tRNA^Lys, the excess tRNA^Lys helped to maintain the concentration of charged-tRNA^Lys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNA^Lys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNA^Lys and the causes of tRNA^Lys starvation in pth(Ts) strains grown at non-permissive temperatures are considered.     

Effect of Flagella on Initial Attachment of Listeria monocytogenes to Stainless Steel

At 22°C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37°C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.     

Heat-shock proteins are associated with hnRNA in Drosophila melanogaster tissue culture cells

Ribonucleoprotein complexes of Drosophila melanogaster Kc tissue culture cells grown at 24°C or heat-shocked at 37°C were cross-linked in vivo by u.v. irradiation. Cross-linked heterogeneous nuclear ribonucleoprotein (hnRNP) complexes were fractionated by oligo(dT)-cellulose chromatography and CsCI density centrifugation. The hnRNP complexes of both 24°C and 37°C culture cells possess buoyant densities in CsCI between = 1.38 g/cm^-3 and 1.43 g/cm^-3. The ³⁵S-labelled proteins bound to the hnRNA of 37°C culture cells correspond in mol. wt. to the so-called heat-shock proteins of 70 K, 68 K, 27 K, 26 K, 23 K and 22 K. The 70 K and 68 K proteins are also present in hnRNP complexes of 24°C culture cells. In addition, several other Drosophila hnRNPs of 140 K, 56 K, 45 K, 43 K, 38 K, 37 K and 34 K, whose synthesis is strongly repressed under heat-shock conditions, could be identified. The results demonstrate that the so-called heat-shock proteins possess a function as RNPs.     

The Isolation of Mms- and Histidine-Sensitive Mutants in NEUROSPORA CRASSA

A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two others were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25° than at 37°. In contrast, mycelial growth is more resistant to MMS at 37°. The possibility of qualitatively different repair processes at these two temperatures is discussed.     

Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

Effect of Butanol Challenge and Temperature on Lipid Composition and Membrane Fluidity of Butanol-Tolerant Clostridium acetobutylicum

The effect of butanol challenge (0, 1.0, 1.5% [vol/vol]) and growth temperature (22, 37, 42°C) on the membrane composition and fluidity of Clostridium acetobutylicum ATCC 824 and a butanol-tolerant mutant, SA-2, was examined in chemically defined medium. Growth of strain ATCC 824 into the stationary phase coincided with a gradual increase in the percent saturated to percent unsaturated (SU) fatty acid ratio. When challenged with butanol at 22 and 37°C, ATCC 824 demonstrated an immediate (within 30 min) dose-response increase in the SU ratio. This strain showed little additional change over a 48-h fermentation. Compared with ATCC 824, growth of SA-2 into the late stationary phase at 22 or 37°C resulted in an overall greater increase in the SU ratio for both unchallenged and challenged cells. This effect was minimized when SA-2 was challenged at 42°C, probably due to the combination of the membrane fluidizing effect of butanol and the elevated temperature. Growth at 42°C resulted in an increase in longer acyl chain fatty acids at the expense of shorter acyl chains for both strains. The membrane fluidity exhibited by SA-2 remained essentially constant at various butanol challenge and temperature combinations, while that for the ATCC 824 strain increased with increasing butanol challenge. By synthesizing an increased amount of saturated fatty acids, the butanol-tolerant SA-2 strain has apparently developed a mechanism for maintaining a more stable membrane environment. Growth of the microorganism is necessary for butanol to fluidize the membrane. Incorporation of exogenous fatty acids (18:1) did not significantly improve the butanol tolerance of either strain. Since SA-2 was able to produce only trace amounts of either butanol or acetone, increased tolerance to butanol does not necessarily coincide with greater solvent yields in this strain.     

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis.

We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.     

Aberrant Forms of Streptococcus cremoris HP

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37°C or in the presence of penicillin. Serial plating and incubation at 37°C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25°C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30°C were transitional phase variants but at 37°C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.     

A Candida albicans Temperature-Sensitive cdc12-6 Mutant Identifies Roles for Septins in Selection of Sites of Germ Tube Formation and Hyphal Morphogenesis

Septins were identified for their role in septation in Saccharomyces cerevisiae and were subsequently implicated in other morphogenic processes. To study septins in Candida albicans hyphal morphogenesis, a temperature-sensitive mutation was created that altered the C terminus of the essential Cdc12 septin. The cdc12-6 cells grew well at room temperature, but at 37°C they displayed expected defects in septation, nuclear localization, and bud morphogenesis. Although serum stimulated the cdc12-6 cells at 37°C to form germ tube outgrowths, the mutant could not maintain polarized hyphal growth and instead formed chains of elongated cell compartments. Serum also stimulated the cdc12-6 mutant to induce a hyphal reporter gene (HWP1-GFP) and a characteristic zone of filipin staining at the leading edge of growth. Interestingly, cdc12-6 cells shifted to 37°C in the absence of serum gradually displayed enriched filipin staining at the tip, which may be due to the altered cell cycle regulation. A striking difference from the wild type was that the cdc12-6 cells frequently formed a second germ tube in close proximity to the first. The mutant cells also failed to form the diffuse band of septins at the base of germ tubes and hyphae, indicating that this septin band plays a role in preventing proximal formation of germ tubes in a manner analogous to bud site selection. These studies demonstrate that not only are septins important for cytokinesis, but they also promote polarized morphogenesis and selection of germ tube sites that may help disseminate an infection in host tissues.     

Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE-homologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli, did not differ from that of the parent strain. When 175 μM extrinsic H₂O₂ was added to the culture medium, bacterial growth of the ΔkatE1 strain was delayed and growth of the ΔkatE1 ΔkatE2 and ΔkatE1 ΔahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1 strain to the inhibition of growth by H₂O₂ was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H₂O₂ stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ΔkatE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H₂O₂ during logarithmic growth and that the function of these genes was influenced by incubation temperature.