Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV _o (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F _(v). For a given voltage,F _(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.     

柱霉属一新种——蝙蝠蛾柱霉

从青海省化隆地区采集冬虫夏草新鲜标本,经分离培养获一纯种,并经鉴定是隶属暗色孢科Dematiaceae的柱霉属Scytalidium Pesante,其形态特征与近似种——黄棕柱霉Scytalidium fulvum进行了比较,由于分生孢子量度及生活习性与黄棕柱霉有着明显差别,故定为一新种,命名为蝙蝠蛾柱霉Scytalidium hepiali C.L.Li sp.nov.该新种在PDA培养基上生长迅速,25℃,培养10天菌落直径2.5cm。菌丝相互平排列成菌丝束。产孢细胞裂殖产孢,节孢子2型:(1)无色,透明,薄壁,圆柱形,桶形。(2)淡褐色,厚壁,椭圆形,桶形,亚球形。蝙蝠蛾柱霉在蛋白胨、葡萄糖液体培养基中进行深层培养,26℃,20天。发酵液用乙醇抽提,减压蒸馏,甲醇溶解,过滤,减压浓缩,蒸馏水溶解,冷冻干燥。从1升发酵液中获虫草菌素制品50～70mg。从拮抗试验表明虫草菌素能抑制枯草芽孢杆菌的生长。     

虫生真菌双生座壳孢的研究

双生座壳孢(Aschersonia duplex Berk.)系日本卷毛蚧的病原真菌。此菌在人工培养基和自然基物上生长好,并能产生大量分生孢子。对 C 源中的蔗糖、葡萄糖、甘露醇、山梨糖;N 源中的蛋白胨,天冬素、L-酪氨酸、KNO_3利用好。生长适温22—26℃。病原流行季节相对湿度在80％以上。田间接种,对寄主致病性强,有应用潜力。     

鹅掌楸种子和胚胎发育的研究

应用控制授粉、软 X-射线法、常规石蜡制片法和荧光检测等手段,研究了鹅掌楸(Lirio-dendron chinense(Hemsl.)Sarg.胚胎发育和控制授粉与结籽率的相关性。控制授粉后2小时花粉萌发,6小时萌发率最高,柱头可授期持续30小时左右。花粉管借助于柱头毛之间的分泌物进入柱头沟,经花柱沟、珠孔塞和珠心冠原进入胚囊,行珠孔受精。授粉后2周,胚乳为2至3细胞厚的狭组织;第6周,胚乳充满胚囊腔,珠心随之解体殆尽;第7到8周,球形胚、心形胚发生;第14到16周,子叶形成;第22周种子或熟,胚乳丰富。单株自然授粉结籽率不足1%。控制授粉后,单个聚合果的最高结籽率可达39%,9个聚合果的平均结籽率为17.7%。     

若干种枝角类卵鞍表面亚显微结构的比较研究

以扫描电镜对发头裸腹溞、蚤状溞、隆线溞指名亚种以及隆线溞东湖一亚种的卵鞍分别进行了比较研究,发现卵鞍表面的亚显微结构有明显的差异,但亚种之间差异较小。隆线溞指名亚种与隆线溞东湖一亚种二者的卵鞍在光学显微镜下看不出有什么差异,然而亚显微结构却互不相同。这就为东湖一新亚种的确定提供了一项可靠的依据,同时也进一步证实卵鞍的超微结构确可作为枝角类分类的表征。     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.     

Use of an organic buffer for the selection of acid tolerantRhizobium meliloti strains

Summary Nine media used to grow rhizobia were examined for their ability to maintain a stable low pH during the growth ofR. meliloti Large fluctuations in the pH of all media were recorded within 72 h, indicating their unsuitability for use in the selection of acid tolerant rhizobia. Morpholino-ethanesulphonic acid (MES) was assessed for its ability to buffer the pH of the media whilst still permitting rapid growth ofR. meliloti, R. trifolii, andBr. lupini. With 30.7 mM MES, the pH of a defined medium containing galactose, arabinose, and glutamate did not change from the initial value of 5.5 even though rhizobial numbers increased from 10⁴ to 10⁹ cells.ml^–1. Even at a buffer concentration of 15.3 mM, pH only increased from 5.5 to 5.6. There was no effect of the buffer on rhizobial growth.     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine