Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.

Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

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)).

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)).

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergDelta mutants, the erg2Delta mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Delta mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D(w)) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC(50) of cycloheximide was closely correlated with S/D(w) in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Delta strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.

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.

Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system.

Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 A resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2(1), with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 A, beta = 104.1 degrees. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.3 A3 Da(-1) and the solvent content is 47%.

New type of pressurized cultivation method providing oxygen for piezotolerant yeast.

For efficient oxygen supply to pressurized culture, we developed a method using a highly pressurized membrane reactor with an air-saturated medium circulation system. The new method increased the cell growth of aerobic yeast approximately 20 folds larger than that in the case of using a conventional method.

Overexpression of Sna3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1.

Tryptophan permease Tat2 in Saccharomyces cerevisiae undergoes Rsp5-dependent degradation upon exposure to high hydrostatic pressure and it limits the growth of tryptophan auxotrophs. Overexpression of SNA3 encoding an endosomal/vacuolar protein possessing the PPAY motif allowed growth at 25 MPa, which was potentiated by marked stabilization of Tat2. This appeared to depend on the PPAY motif, which interacted with the WW domain of Rsp5. Subcellular localization of Rsp5 was unchanged by overexpression of either SNA3 or SNA3-AAAY. While the loss of Bul1, a binding protein of Rsp5, or the rsp5-ww3 mutation allowed high-pressure growth, overexpression of BUL1 abolished the Sna3-mediated growth at 25 MPa. These results suggest that Sna3 and Bul1 compete for the WW domain of Rsp5 upon Tat2 ubiquitination.

Sorting defects of the tryptophan permease Tat2 in an erg2 yeast mutant.

Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, 'lipid rafts,' which are thought to be critical for intracellular protein sorting in eukaryotic cells. When the activity of Erg9 involved in the first step of ergosterol biogenesis, but not that of Erg6 involved in a late step, is compromised, vacuolar degradation of the tryptophan permease Tat2 is promoted. It is unknown whether this difference simply reflects the difference between the inhibition of early and late steps. Here, it is shown that the deletion in ERG2, which encodes sterol C8-C7 isomerase (the next enzymatic step after Erg6), promotes the vacuolar degradation of Tat2. It suggests that the accumulation of specific sterol intermediates may alter lipid raft structures, promoting Tat2 degradation. The erg2Delta-mediated Tat2 degradation required Tat2 ubiquitination. Lipid raft association of Tat2 is compromised in erg2Delta cells. The erg2Delta mutation showed a synthetic growth defect with the trp1 mutation, indicating that Tat2 sorting is preferentially compromised in these mutants. Consistent with this notion, the raft-associated protein Pma1 was associated with detergent-resistant membranes and sorted to the plasma membrane. This study suggests the potential for the pharmacological control of cellular nutrient uptake in humans by regulating enzymes involved in cholesterol biogenesis.

Crystal structures of the clock protein EA4 from the silkworm Bombyx mori.

Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called "peptidyl inhibitory needle") under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock.

Crystal structures of unliganded and half-liganded human hemoglobin derivatives cross-linked between Lys 82beta1 and Lys 82beta2.

A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.