Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus: ROLE IN COMPLEX STABILITY,PILIATION, ADHESION,TWITCHING MOTILITY,AND NATURAL TRANSFORMATION*

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.     

Identification and characterization of a unique,zinc-containing transport ATPase essential for natural transformation in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27

Thermus thermophilus is a model strain to unravel the molecular basis of horizontal gene transfer in hot environments. Previous genetic studies led to the identification of a macromolecular transport machinery mediating DNA uptake in an energy-dependent manner. Here, we have addressed how the transporter is energized. Inspection of the genome sequence revealed four putative transport (AAA) ATPases but only the deletion of one, PilF, led to a transformation defect. PilF is similar to transport ATPases of type IV and type II secretions systems but has a unique N-terminal sequence that carries a triplicated GSPII domain. To characterize PilF biochemically it was produced in Escherichia coli and purified. The recombinant protein displayed NTPase activity with a preference for ATP. Gel filtration analyses combined with dynamic light scattering demonstrated that PilF is monodispersed in solution and forms a complex of 590 ± 30 kDa, indicating a homooligomer of six subunits. It contains a tetracysteine motif, previously shown to bind Zn²⁺ in related NTPases. Using atomic absorption spectroscopy, indeed Zn²⁺ was detected in the enzyme, but in contrast to all known zinc-binding traffic NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn²⁺. Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation.     

PilF is an outer membrane lipoprotein required for multimerization and localization of the Pseudomonas aeruginosa Type IV pilus secretin

Type IV pili (T4P) are retractile appendages that contribute to the virulence of bacterial pathogens. PilF is a Pseudomonas aeruginosa lipoprotein that is essential for T4P biogenesis. Phenotypic characterization of a pilF mutant confirmed that T4P-mediated functions are abrogated: T4P were no longer present on the cell surface, twitching motility was abolished, and the mutant was resistant to infection by T4P retraction-dependent bacteriophage. The results of cellular fractionation studies indicated that PilF is the outer membrane pilotin required for the localization and multimerization of the secretin, PilQ. Mutation of the putative PilF lipidation site untethered the protein from the outer membrane, causing secretin assembly in both inner and outer membranes. T4P-mediated twitching motility and bacteriophage susceptibility were moderately decreased in the lipidation site mutant, while cell surface piliation was substantially reduced. The tethering of PilF to the outer membrane promotes the correct localization of PilQ and appears to be required for the formation of stable T4P. Our 2.0-Å structure of PilF revealed a superhelical arrangement of six tetratricopeptide protein-protein interaction motifs that may mediate the contacts with PilQ during secretin assembly. An alignment of pseudomonad PilF sequences revealed three highly conserved surfaces that may be involved in PilF function.     

Conformational analysis by nuclear magnetic resonance: insulin

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).     

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ.     

A solution equivalent of the 2Zn----4Zn transformation of insulin in the crystal

Circular dichroic spectroscopy clearly reveals a solvent-induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn----4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B-chain N-terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn----4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+). It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des-pentapeptideB26-30-insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off-axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1-B29-cross-linked insulin in spite of the partially reduced flexibility.     

The polymerization pattern of zinc(II)-insulin at pH 7.0.

Sedimentation equilibrium experiments were conducted at pH 7.0 using solutions of bovine insulin containing 2 mol of zinc(II) ions per six base-mol of insulin. A detailed analysis of these results revealed the existence of a stable zinc-insulin hexamer together with linked polymerization reactions. Specifically these are a background polymerization of zinc-free insulin as previously described by Jeffrey et al. ((1976) Biochemistry 15, 4660--4665) and a slight tendency for the zinc-insulin hexamer to undergo indefinite self-association. Equilibrium constants governing these reactions are reported together with equations which permit calculation of the composition of the solution at any given total concentration. Comment is made on the possible biological significance of this linked polymerization pattern, and on the likely identity of the structure of the stable zinc-insulin hexamer with that previously reported from X-ray crystallographic studies.     

Type IV Pilus Biogenesis,Twitching Motility,and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF,PilT1, and PilT2

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.     

ZnT-8, A Pancreatic Beta-Cell-Specific Zinc Transporter

The zinc content in the pancreatic beta cell is among the highest of the body. Zinc appears to be an important metal for insulin-secreting cells as insulin is stored inside secretory vesicles as a solid hexamer bound with two Zn²⁺ ions per hexamer. Zinc is also an important component of insulin secretion mechanisms and is likely to modulate the function of neighbouring cells via paracrine/autocrine interactions. Therefore beta cells undoubtedly need very efficient and specialized transporters to accumulate sufficient amounts of zinc in secretion vesicles. We report here the discovery and the characteristics of a new zinc transporter, ZnT-8, belonging to the CDF (Cation Diffusion Facilitator) family and expressed only in pancreatic beta cells. This transporter, localized in secretion vesicles membrane, facilitates the accumulation of zinc from the cytoplasm into intracellular insulin-containing vesicles and is a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta cells. We discovered mammalian orthologs (rat, mouse, chimpanzee, and dog) and found these ZnT-8 proteins very similar (98% conserved amino acids) to human ZnT-8, indicating a high conservation during evolution.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Influence of Zn2+ and Fe3+ on polysaccharide production and mycelium/yeast dimorphism of Aureobasidium pullulans in batch cultivations

Cultivation of Aureobasidium pullulans in medium with a low concentration of yeast extract (0.4 g/l) led to a decrease in the growth rate early in the fermentation as compared to cultivations in medium with high concentration of yeast extract. When this medium was supplemented with zinc and iron the cultivation closely resembled that obtained in medium with high concentration of yeast extract (4.0 g/l). The culture retained a high growth rate throughout the fermentation and the initiation of the mycelial to yeast (M-Y) transition and the exopolysaccharide production was delayed. In a defined medium or in defined medium without iron only a little exopolysaccharide was produced and the yeast fraction of the total biomass at the onset of the stationary phase was 22%–25%. However, cultivation in the defined medium without zinc resulted in a high production of exopolysaccharide and an increased intensity of the M-Y transition, which led to a yeast fraction of 41%.     

An electron paramagnetic resonance study of native and modified freeze-dried cupric insulin hexamer.

Cupric insulin was modified by the addition of cross-linking disulphide bridges between hexamers. The electron paramagnetic resonance (EPR) spectrum of this freeze-dried material was compared with that of freeze-dried unmodified cupric insulin containing various amounts of copper and added water. The modified insulin was found to have cupric ion sites magnetically very similar to that of native insulin containing two cupric ions per hexamer. Native hexamer produced in the presence of 2 Cu(II) ions per hexamer gave, after freeze-drying, an EPR spectrum with A_∥^Cu=16.5 mT, g_∥=2.285 and g_⊥=2.059 (site 1). The use of 4 or 6 Cu(II) ions per hexamer resulted in spectra with two components-a major component with the same A_∥^Cu and g_∥ values as the sample containing 2 Cu(II) ions (site 1) and an additional minor component (site 2). These sites have been identified with the analogous zinc binding site within the hexamer formed by three B-10 histidine residues (site 1) [1, 2] and the site formed by the B-1 α-amino and A-17 glutamyl-γ-barboxylic acid functions where excess zinc is bound (site 2) [3, 4]. The addition of water to native hexamer containing 2, 4, or 6 Cu(II) ions resulted in the appearance of three distinct EPR absorptions, one of which had the same parameters as the freeze-dried native insulin containing 2 Cu(II) ions per hexamer (site 1). Two further sites appeared (3 and 4) with the following parameters: A_∥^Cu=15.0 mT, g_∥=2.353, and g_⊥=2.07; A_∥^Cu=16.5 mT, g_∥=2.315, and g_⊥=2.07, respectively.     

Structural transition in the metal-free hexamer of protein-engineered [B13 Gln]insulin

For hexamer formation of native insulin the repulsive potential of six B13 Glu carboxylate groups coming together in the centre is overcome by zinc binding to B10 His. Substitution of Gln for Glu in position B13 by site-directed mutagenesis, i.e. replacement of the repelling carboxylates by amide groups, which are offering H-bonding potential, enhances association and allows a metal-free hexamer to form. Merely upon addition of zinc ions this hexamer undergoes the T6----T3R3 respectively T6----R6 structural transition which in the native 2Zn insulin hexamer is inducible only by additives like inorganic anions or phenolic compounds. [B13 Gln]Insulin hexamers are transformed by phenolic compounds, but not by anions, even in the absence of any metal. The structural transformation of insulin can thus be brought about in two ways: By inorganic ions with the zinc ions as their points of attack, which preexist in the nontransformed hexamer, and by phenol, for which the binding sites close to the B5 histidines come into existence only with the transformation. Therefore transformed and non-transformed hexamers, i.e. molecules with helical and extended B chain N-terminus, must be related in a dynamic equilibrium. Phenol acts as a wedge jamming the structure in the transformed state and trapping the zinc ions. Combination of transformed 2Zn[B13 Gln]insulin and metal-free native insulin in the absence of additives results in a redistribution of the zinc ions in favour of native insulin which is an outcome of the dynamic equilibrium and also demonstrates an influence of B13 charge on metal binding affinity. Transformation of a single subunit in a hexamer would lead to bad contacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetate as Sole Carbon and Energy Source for Growth of Methanosarcina Strain 227

Methanosarcina strain 227 grew rapidly and produced methane on a mineral medium containing acetate as the sole added organic substrate. Cell yields but not doubling times were affected by the presence or absence of yeast extract. Greater cell yields occurred in yeast extract medium than in mineral medium. Radioactive labeling studies showed that acetate was decarboxylated in mineral medium, as was shown previously in complex medium. The specific radioactivity of methane produced per specific acitvity of acetate added was not significantly different in yeast extract medium compared with mineral medium. Unequivocal evidence indicates that the cleavage of acetate to methane and carbon dioxide provided the energy for growth in the presence or absence of other organic compounds; these latter compounds do not serve as energy sources, electron donors, or significant sources of methane during this aceticlastic reaction.     

Growth media effects on morphology and 17β-HSD activity in the fungusCurvularia lunata

The filamentous fungusCurvularia lunata has enzymatic activities of biotechnological significance. Its 11β-hydroxylase actvity produces hydrocortisone, and its 17-ketosteroid reductase (17β-HSD) activity is important in the production of 17β-hydroxysteroids. We have examined the effects of different media on the 17β-HSD activity, and on the morphology and ultrastructure ofC. lunata in the search for a correlation between high enzymatic activity, morphology and ultrastructure. The highest 17β-HSD activity per dry weight ofC. lunata was seen in Czapek medium, while more product per volume was formed in malt extract broth. In all growth media tested, the highest 17β-HSD activity coincided with the exponential phase of growth. In Czapek broth medium, fungal hyphae formed pellets, while in malt extract and yeast nitrogen base broth media, the fungus formed branched hyphae dispersed throughout the media. Scanning electron microscopy revealed differences in the thickness of hyphal cells; those of the fungus grown in Czapek and yeast nitrogen base media were much thicker than those in malt extract medium. Transmission electron microscopy showed melanized cell walls in the fungus grown in malt extract and Czapek media, but not in yeast nitrogen base medium. Our results show that theC. lunata with the highest 17β-HSD activity grow as dispersed, melanized mycelia with thin hyphae.     

Chemical composition and biological action of rowanberry extract

The object of this study was the extract from the dried pomace of the Amur rowanberry (Sorbus amurensis Koehne) after the juice separation. The content of total polyphenols, lipid composition, and antiradical activity of the extract were studied. The total content of polyphenols proved to be 80 ± 2,5 mg-equivalents of gallic acid per 100 mL of the extract, suggesting the high level of its antiradical activity (29.73 ± 0.23 mmolequivalents of trolox per liter). The lipid part of the extract included three phospholipid fractions and six fractions of neutral lipids. Their content was found to be 13.4 ± 0.14 mg of total lipids/mL. These results pointed to a high biological activity of the extract which could be considered as a promising object for further experimental studies. The Amur rowanberry extract was shown to exhibit a pronounced antioxidant and hepatoprotective activity on the laboratory model of the carbon-tetrachloride-induced hepatitis in rats with endogastric administration at a dose of 100 mg of total polyphenols per kg of body weight. Administration of the extract resulted in a decrease in the content of total lipids, specific liver weight, and the activity of alanine aminotransferase. The level of the reduced glutathione and the activity of antioxidant and lysosomal enzymes were also normalized. The rowanberry extract seemed to be a promising source for the creation of biologically active food supplements with high antioxidant and antiradical activity.     

The zinc content of yeast alcohol dehydrogenase

Analyses for zinc in high specific activity preparations of yeast alcohol dehydrogenase (YADH) indicate a metal content of 1.8–1.9 moles of zinc per mole of enzyme subunit. This zinc content is observed for YADH prepared from Bakers yeast by recrystallization from Am₂SO₄ containing 1 mM EDTA, followed by chromatography on DE-52 and Sephadex-G-200. YADH obtained from Boehringer-Mannheim is characterized by a variable specific activity: preparations with Sp. Ac. = 380–400 U/mg contain 1.8–1.9 moles of zinc per mole of subunit. Dialysis of YADH against EDTA (pH 8.5, 25°, under N₂) reduces the specific activity and zinc content in an approximately linear fashion down to a Sp. Ac. = 150 U/mg, consistent with the preferential loss of a single, weakly bound zinc per subunit which is essential for catalytic activity. Dialysis of YADH against 1 mM ZnCl₂ (pH 6.5–8.5, 25°, under N₂) does not lead to an increase in the zinc content of the enzyme, indicating that under these conditions zinc does not bind adventitiously to YADH. Dialysis against 50 mM CoSO₄ (pH 5.5, 25°, under N2, 60–90 hr) leads to an exchange of ≈ 40% of the enzyme-bound zinc by cobalt. Our preparations of YADH are consistently characterized by a zinc content of ≈ 2 per subunit and we are unable to reduce the zinc content of YADH by dialysis against EDTA without a concomitant loss in enzyme activity, in contrast to reports of one zinc per subunit [Veillon, C. and Sytkowski, A.J., BBRC $\text{67}$: 1499 (1975); Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA $\text{41}$: 327 (1955)]. The findings reported here, together with the observed structural similarities between YADH and horse liver alcohol dehydrogenase [Jornvall, H., Woenckhaus, C. and Johnscher, G., Eur. J. Biochem. $\text{53}$: 71 (1975)], suggest a role for zinc at both a structural and catalytic site in YADH.     

Isoenzymes of transaldolase in strains of Candida utilis and effect of different growth conditions

The isozymes of transaldolase have been investigated in six strains of Candida utilis. Four of these strains were cloned and in three of them more than one isozyme was found, indicating that mutltiple forms of the activity are present in the same cell. Cloned cells of one strain (C0U) were grown on three different media (YED, 1% dextrose, 1% yeast extract; YEX, 1% xylose, 1% yeast extract; AM, 1% dextrose, 1% ammonium sulfate, 0.025% yeast extract plus mineral salts) and analyzed for transaldolase isozyme patterns. Without autolysis the homogenates showed identical profiles with a single peak of activity characteristic of isozyme I. In spite of significantly different levels of pentose-phosphate pathway metabolites expected these patterns were not appreciably altered. Our data reveal a characteristic pattern for each strain and do not indicate a ready alteration of transaldolase isozymes as a response to changes in nutritional conditions.     

A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis

Zinc deficiency in dark-grown Euglena gracilis Klebs, Z strain Pringsheim, results in the disappearance of cytoplasmic ribosomes. In contrast, ribosomes in zinc-sufficient Euglena are conserved, do not undergo turnover, and can be demonstrated at any stage of growth. The zinc content of ribosomes from zinc-deficient Euglena just prior to ribosomal disappearance is 300 to 380 micrograms of zinc per gram rRNA as compared to 650 to 1280 micrograms of zinc per gram rRNA in ribosomes from zinc-sufficient cells. Ribosomal disappearance is believed to involve a generalized disintegration process related to the lower content of zinc in the ribosomes. Reappearance of ribosomes requires the addition of zinc. It is proposed that adequate zinc may be essential for normal tertiary and quaternary structure of the cytoplasmic ribosomes of Euglena.     

Accumulation and cellular distribution of zinc by brewing yeast

This study focused on the interactions between yeast and zinc in relation to beer fermentations. Yeast accumulation of zinc from growth media, including malt wort, was found to be rapid following inoculation with a brewing strain of Saccharomyces carlsbergensis. In contrast, at the onset of the fermentation, the uptake of other divalent cations such as magnesium and calcium was not as pronounced compared with zinc. At the end of fermentation, both growth media and yeast cells became zinc-depleted, the latter due to dilution of zinc to daughter cells following growth and cell division. In addition, in brewing fermenters, the levels of intracellular zinc were much higher in suspended yeast cells compared with cells that sedimented in the yeast cone at the end of fermentation. This may result in impaired yeast performance in subsequent fermentations if yeast is recycled into low zinc media and if the sub-population is composed by zinc-depleted daughter cells. Cellular uptake of zinc was mediated by a metabolism-dependent mechanism as evidenced by impaired uptake following heat shock. Zinc was thereafter localised in the yeast cell vacuole. As industrial fermentation processes may occasionally be suppressed due to zinc deficiencies, the findings of this study are pertinent for several yeast-based industries, especially beer production.