Keiko Usui

Fluconazole Modulates Membrane Rigidity, Heterogeneity, and Water Penetration into the Plasma Membrane in Saccharomyces cerevisiae

Azole anitifungal drugs such as fluconazole inhibit 14alpha-demethylase. The mechanism of fluconazole action on the plasma membrane is assumed to be ergosterol depletion and accumulation of a toxic sterol, 14alpha-methyl-3,6-diol, that differs in C-6 hydroxylation, B-ring saturation, C-14 methylation, and side-chain modification. Nevertheless, little is known about how these sterol modifications mechanically influence membrane properties and hence fungal viability. Employing time-resolved measurement with a fluorescence anisotropy probe, 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), we demonstrated that fluconazole administration decreased the rigidity of the plasma membrane of Saccharomyces cerevisiae, leading to a dramatic reduction in the order parameter (S) from 0.965 to 0.907 and a 5-fold acceleration of the rotational lipid motion. This suggests that the altered sterol has a deleterious impact on membrane packing, resulting in increased fluidity. Deletion of ERG3 confers hyperresistance to fluconazole by circumventing the accumulation of 14alpha-methyl-3,6-diol and instead produces 14alpha-methylfecosterol lacking the 6-OH group. We found that ERG3 deletion mitigated the fluconazole-induced loss of membrane rigidity with S remaining at a higher value (=0.922), which could contribute to the fluconazole resistance in the erg3Delta mutant. The reduced ability of the 6-OH sterol to stiffen lipid bilayers was supported by the finding that 30 mol % of 6alpha-hydroxy-5alpha-cholestanol marginally increased the S value of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes, while cholesterol and dihydrocholesterol markedly increased it. The decay of the TMA-DPH fluorescence was bimodal in the wild-type strain. This heterogeneity could have arisen from varying degrees of water penetration into the plasma membrane. Fluconazole eliminated the heterogeneity of the dielectric characteristic of the membrane interfacial region, and concomitantly the TMA-DPH lifetime was shortened. Therefore, we conclude that 14alpha-methyl-3,6-diol is insufficient to pack the plasma membrane, allowing water penetration, which is consistent with membrane disorder after fluconazole administration. Our findings illustrate the role of ergosterol in maintaining membrane heterogeneity and preventing water penetration as well as maintaining the rigidity of the plasma membrane interfacial region.

Eicosapentaenoic acid plays a role in stabilizing dynamic membrane structure in the deep-sea piezophile Shewanella violacea: A study employing high-pressure time-resolved fluorescence anisotropy measurement

Shewanella violacea DSS12 is a psychrophilic piezophile that optimally grows at 30MPa. It contains a substantial amount of eicosapentaenoic acid (EPA) in the membrane. Despite evidence linking increased fatty acid unsaturation and bacterial growth under high pressure, little is known of how the physicochemical properties of the membrane are modulated by unsaturated fatty acids in vivo. By means of the newly developed system performing time-resolved fluorescence anisotropy measurement under high pressure (HP-TRFAM), we demonstrate that the membrane of S. violacea is highly ordered at 0.1MPa and 10°C with the order parameter S of 0.9, and the rotational diffusion coefficient D(w) of 5.4μs(-1) for 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene in the membrane. Deletion of pfaA encoding the omega-3 polyunsaturated fatty acid synthase caused disorder of the membrane and enhanced the rotational motion of acyl chains, in concert with a 2-fold increase in the palmitoleic acid level. While the wild-type membrane was unperturbed over a wide range of pressures with respect to relatively small effects of pressure on S and D(w), the ΔpfaA membrane was disturbed judging from the degree of increased S and decreased D(w). These results suggest that EPA prevents the membrane from becoming hyperfluid and maintains membrane stability against significant changes in pressure. Our results counter the generally accepted concept that greater fluidity is a membrane characteristic of microorganisms that inhabit cold, high-pressure environments. We suggest that retaining a certain level of membrane physical properties under high pressure is more important than conferring membrane fluidity alone.

Brevundimonas abyssalis sp. nov., a dimorphic prosthecate bacterium isolated from deep-subsea floor sediment

A novel Gram-negative, aerobic, psychrotolerant, alkali-tolerant, heterotrophic and dimorphic prosthecate bacterium, designated strain TAR-001(T), was isolated from deep-sea floor sediment in Japan. Cells of this strain had a dimorphic life cycle and developed an adhesive stalk at a site not coincident with the centre of the cell pole, and the other type of cell, a swarm cell, had a polar flagellum. Colonies were glossy, viscous and yellowish-white in colour. The temperature, pH and salt concentration range for growth were 2-41 °C, pH 6.5-10.0 and 1-4% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-001(T) belongs to the family Caulobacteraceae of the class Alphaproteobacteria, and lies between the genus Brevundimonas and the genus Caulobacter. Levels of similarity between the 16S rRNA gene sequence of strain TAR-001(T) and those of the type strains of Brevundimonas species were 93.3-95.7%; highest sequence similarity was with the type strain of Brevundimonas diminuta. Levels of sequence similarity between those of the type strains of Caulobacter species were 94.9-96.0%; highest sequence similarity was with the type strain of Caulobacter mirabilis. The G+C content of strain TAR-001(T) was 67.6 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C18:1ω7c and C16:0, and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1→4)-α-D-glucopyranuronosyl]glycerol suggests strain TAR-001(T) is more closely to the genus Brevundimonas than to the genus Caulobacter. The mean DNA-DNA hybridization levels between strain TAR-001(T) and the type strains of two species of the genus Brevundimonas were higher than that of the genus Caulobacter. On the basis of polyphasic biological features and the 16S rRNA gene sequence comparison presented here, strain TAR-001(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas abyssalis sp. nov. is proposed; the type strain is TAR-001(T) (=JCM 18150(T)=CECT 8073(T)).

Brevundimonas denitrificans sp. nov., a denitrifying bacterium isolated from deep subseafloor sediment

A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002(T), was isolated from deep seafloor sediment in Japan. Colonies lacked lustre, and were viscous and translucent white. The ranges of temperature, pH and salt concentration for growth were 8-30 °C, pH 6.0-10.0 and 1-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-002(T) belongs to the genus Brevundimonas of the class Alphaproteobacteria. Levels of similarity between the 16S rRNA gene sequence of strain TAR-002(T) and those of the type strains of species of the genus Brevundimonas were 93.5-98.9%; the most closely related species was Brevundimonas basaltis. In DNA-DNA hybridization assays between strain TAR-002(T) and its phylogenetic neighbours, Brevundimonas lenta DS-18(T), B. basaltis J22(T), Brevundimonas subvibrioides ATCC 15264(T) and Brevundimonas alba DSM 4736(T), mean hybridization levels were 6.4-27.7%. The G+C content of strain TAR-002(T) was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C(18:1)ω7c and C(16:0), and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1 → 4)-α-D-glucopyranuronosyl]glycerol (DGL) indicates the affiliation of strain TAR-002(T) with the genus Brevundimonas. On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas denitrificans sp. nov. is proposed; the type strain is TAR-002(T) ( =NBRC 110107(T) =CECT 8537(T)).

Polycladomyces abyssicola gen. nov., sp. nov., a thermophilic filamentous bacterium isolated from hemipelagic sediment

A novel filamentous bacterium, designated strain JIR-001(T), was isolated from hemipelagic sediment in deep seawater. This strain was non-motile, Gram-positive, aerobic, heterotrophic and thermophilic; colonies were of infinite form and ivory coloured with wrinkles between the centre and the edge of the colony on ISP2 medium. The isolate grew aerobically at 55-73 °C with the formation of aerial mycelia; spores were produced singly along the aerial mycelium. These morphological features show some similarities to those of the type strains of some species belonging to the family Thermoactinomycetaceae. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain JIR-001(T) belongs to the family Thermoactinomycetaceae within the class Bacilli. Similarity levels between the 16S rRNA gene sequence of strain JIR-001(T) and those of the type strains of Thermoactinomycetaceae species were 85.5-93.5%; highest sequence similarity was with Melghirimyces algeriensis NariEX(T). In the DNA-DNA hybridization assays between strain JIR-001(T) and its phylogenetic neighbours the mean hybridization levels with Melghirimyces algeriensis NariEX(T), Planifilum fimeticola H0165(T), Planifilum fulgidum 500275(T) and Planifilum yunnanense LA5(T) were 5.3-7.5, 2.3-4.7, 2.1-4.8 and 2.5-4.9%, respectively. The DNA G+C content of strain JIR-001(T) was 55.1 mol%. The major fatty acids were iso-C15:0, iso-C17:0, iso-C16:0 and C16:0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, glucolipid, phosphatidylserine, an amino-group containing phospholipid, an unknown phospholipid and two unknown lipids. The predominant menaquinone was MK-7 and the cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, strain JIR-001(T) is considered to represent a novel species in a new genus of the family Thermoactinomycetaceae, for which the name Polycladomyces abyssicola gen. nov., sp. nov. is proposed. The type strain of Polycladomyces abyssicola is JIR-001(T) (=JCM 18147(T)=CECT 8074(T)).

Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov., isolated from a decaying bamboo sunken in the marine environment, and emended description of the genus Thalassospira.

Two marine bacteria, designated strains MBE#61(T) and MBE#74(T), were isolated from a piece of sunken bamboo in the marine environment in Japan. Both of these strains were Gram-stain-negative, but had different cell shapes: MBE#61(T) was spiral, whereas MBE#74(T) was rod-shaped. The temperature, pH and salt concentration ranges for growth of strain MBE#61(T) were 4-38 °C (optimal at 32 °C), pH 4.5-11.0 (optimal at pH 7.0-8.0) and 1-11 % (optimal at 2 %) NaCl, whereas those of strain MBE#74(T) were 4-36 °C (optimal at 30 °C), pH 4.0-10.5 (optimal at pH 7.0-8.0) and 1-12 % (optimal at 4 %) NaCl. Phylogenetic analysis based on partial 16S rRNA gene sequences revealed that both strains belong to the genus Thalassospira within the class Alphaproteobacteria. Similarity between the 16S rRNA gene sequence of strain MBE#61(T) and those of the type strains of species of the genus Thalassospira was 97.5-99.0 %, and that of strain MBE#74(T) was 96.9-98.6 %; these two isolates were most closely related to Thalassospira lucentensis QMT2(T). However, the DNA-DNA hybridization values between T. lucentensis QMT2(T) and strain MBE#61(T) or MBE#74(T) were only 16.0 % and 7.1 %, respectively. The DNA G+C content of strain MBE#61(T) was 54.4 mol%, and that of strain MBE#74(T) was 55.9 mol%. The predominant isoprenoid quinone of the two strains was Q-10 (MBE#61(T), 97.3 %; MBE#74(T), 93.5 %). The major cellular fatty acids of strain MBE#61(T) were C18 : 1ω7c (31.1 %), summed feature 3 comprising C16 : 0ω7c/iso-C15 : 0 2-OH (26.1 %) and C16 : 0 (20.9 %); those of strain MBE#74(T) were C16 : 0 (26.2 %), C17 : 0 cyclo (19.9 %) and C18 : 1ω7c (12.1 %). On the basis of these results, strain MBE#61(T) and strain MBE#74(T) are considered to represent novel species of the genus Thalassospira, for which names Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov. are proposed. The type strains are MBE#61(T) ( = JCM 18968(T) = CECT 8273(T)) and MBE#74(T) ( = JCM 18969(T) = CECT 8274(T)), respectively. An emended description of the genus Thalassospira is also proposed.

Effects of high hydrostatic pressure on the dynamic structure of living Escherichia coli membrane: a study using high pressure time-resolved fluorescence anisotropy measurement

High hydrostatic pressure has a significant impact on the structure of biological membranes. Here, we elucidated the effects of hydrostatic pressure on a model palmitoyl-oleylphosphatidylcholine (POPC) membrane and a natural membrane of mesophilic Escherichia coli by means of time-resolved fluorescence anisotropy measurement using 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Hydrostatic pressure of 50 MPa increased the order parameter S of the E. coli cell membrane from 0.782 to 0.815 at 37°C and it markedly reduced the rotational diffusion coefficient D w of TMA-DPH to 69.4% of that observed at 0.1 MPa. High pressure exerted similar effects on the living E. coli cell membrane to those obtained with POPC in terms of the changes in S and D w . Therefore, the dynamic behavior of the natural membrane in response to high pressure could in part be deduced from studies of the model membrane as long as the membrane is in the liquid-crystalline phase.

Aneurinibacillus tyrosinisolvens sp. nov., a tyrosine-dissolving bacterium isolated from organics- and methane-rich seafloor sediment

A novel Gram-positive-staining, strictly aerobic and heterotrophic bacterium, designated strain LL-002T, was isolated from organics- and methane-rich seafloor sediment at a depth of 100 m in Kagoshima Bay, Kagoshima, Japan. Colonies were lustreless and translucent white in colour. The temperature, pH and salt concentration ranges for growth were 10-30 °C, pH 6.0-6.5 and 0-1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-002T belongs to the genus Aneurinibacillus of the family Paenibacillaceae. 16S rRNA gene sequence similarities between strain LL-002T and the type strains of species of the genus Aneurinibacillus were 92.8-95.7 %; the highest sequence identity was with the type strain of Aneurinibacillus migulanus. The DNA G+C content of strain LL-002T was 46.2 mol%. MK-7 was the predominant menaquinone. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and the cell-wall peptidoglycan contained meso-diaminopimelic acid and glutamic acid, glycine and alanine in addition to muramic acid and glucosamine. The peptidoglycan type was A1γ. In DNA-DNA hybridization assays between strain LL-002T and the type strains of the other species of the genus Aneurinibacillus, the level of hybridization was 6.3-30.1 %. On the basis of its biological features and the 16S rRNA gene sequence comparison presented here, strain LL-002T is considered to represent a novel species of the genus Aneurinibacillus, for which the name Aneurinibacillus tyrosinisolvens sp. nov. is proposed; the type strain is LL-002T ( = NBRC 110097T = CECT 8536T).

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp−) strains under pressures of 15–25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp− cells to grow at 15–25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.