Extracellular Accumulation of Phospholipids,UDP-N-Acetylhexosamine Derivatives and L-Glutamic Acid by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin to cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium caused the simultaneous excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid.

Among many antibiotics which inhibit cell wall synthesis, only the inhibitors of peptideglycan transpeptidase such as penicillin G and cephaloridine were effective for inducing the excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid, while the others promoted only the excretion of UDP-N-acetylhexosamine derivatives.

From the close relationship between the excretion of L-glutamic acid and the excretion of phospholipids, it was suggested that the action of penicillins and cephalosporins on the cell membrane resulted in the excretion of L-glutamic acid.     

Relation between Fatty Acid Composition of Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum

Relation between fatty acid composition of cellular phospholipids and the excretion of L-glutamic acid was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When grown on n-hexadecane, the proportion of unsaturated fatty acids was higher in L-glutamic acid-accumulating cells than in L-glutamic acid-nonaccumulating cells. When grown on fructose or acetic acid, the reverse relation was observed. Moreover, cells containing no oleic acid produced L-glutamic acid from n-pentadecane.

These results suggest that the membrane permeability to L-glutamic acid is not always controlled by the cellular content of unsaturated fatty acids.     

Separation and Identification of UDP-N-Acetylhexosamine Derivatives Excreted by Penicillin-treated Corynebacterium alkanolyticum

N-Acetylhexosamine derivatives, which are intermediates of cell wall synthesis, were detected in UV-absorbing substances excreted by penicillin-treated Corynebacterium alkanolyticum. Gel filtration, using Sephadex G-25, separated N-acetylhexosamine derivatives to three components, each of which was purified by Dowex 1 × 2 column and paper chromatographies.

From the analytical studies, N-acetylhexosamine derivatives were found to be composed of UDP-N-acetylmuramic acid-(diaminopimelic acid, glutamic acid, alanine), UDP-N-acetylhexosaminuronic acid and UDP-N-acetylglucosamine.     

Conversion of Citrate to Extracellular Glutamate by Penicillin-treated Resting Cells of Corynebacterium glutamicum

Triggering of glutamate excretion by penicillin is thought to occur by increasing cell permeability. It seemed odd that glucose-grown resting cells, after penicillin treatment, would not convert citrate to extracellular glutamate especially since citrate had been reported to be a substrate for the glutamate fermentation. Citrate was not even taken up by such cells. Upon addition of at least 2 percent glucose, citrate was converted to extracellular glutamate. Both glucose and citrate were used simultaneously and citrate metabolism continued even after sugar was exhausted. It was suspected that glucose was required as energy source for induction of a citrate-transport system. Resting cells pregrown in glucose plus citrate, were indeed found to take up citrate and convert it to extracellular glutamate even in the absence of sugar. In line with the induction hypothesis, chloramphenicol inhibited the metabolism of citrate by glucose-grown resting cells but had no such effect on the citrate-adapted cells. The antibiotic did not inhibit glucose utilization by citrate-adapted or unadapted resting cells.     

Studies on the Polymorphism of L-Glutamic Acid

The growth rate of the α-crystal of L-glutamic acid was measured under various degrees of supersaturation and temperatures. The rate constants and the activation energies for (0 0 1) and (1 1 1) faces were measured and the latter values were 6.7 and 11.5 kcal/mol, respectively. The controlling process of the α-crystal growth was investigated by comparison of Sherwood numbers of dissolution and crystallization, and the crystallization process was found to be controlled by the surface reaction.     

The Relation between Accumulation of Abscisic Acid and Proline in Detached Rice Leaves

The relation between abscisic acid (ABA) and proline accumulation was investigated in detached rice (Oryza sativa L.) leaves. In darkness, proline content increased about 2-, 2,5- and 6-fold after 24, 48 and 72 h. ABA content reached maximum after 48 h. In the light, proline content remained almost unchanged until 48 h and subsequently increased slightly. ABA content in the light was lower than in darkness, but the maximum was also after 48 h. During 12-h exposure to decreased air humidity, proline content gradually increased, but ABA content increased about 25-fold after 4 h and declined thereafter. Exogenous application of ABA resulted in an increase in proline content in detached rice leaves under both light and darkness.     

Studies on the Polymorphism of L-Glutamic Acid

The velocity of the α-β transition was measured in two cases, i.e., when α-crystals stayed in the saturated aqueous solution, and when they were left to stand in an air-bath at various temperatures ranging from 20° to 100°C. And it was deduced from the results of the measurement that this transition is due to the recrystallization of α-crystals into the β-form on the inner surfaces of the crystal and in the bulk of the solution, and the transition on the inner surfaces will be dominant, especially at the initial stage of the transition.     

Intraspecific Variation of Unusual Phospholipids from Corynebacterium spp. Containing a Novel Fatty Acid

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132^T and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132^T and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Effect of L-Aspartic Acid and L-Glutamic Acid on Production of L-Proline

To elucidate the effect of aspartic acid on growth of Kurthia catenaforma during the proline fermentation, this organism was compared with other bacteria with respect to the rate of consumption of aspartic acid, and to the activities of enzymes concerned in the metabolism of aspartic acid. Although no marked difference in enzyme activities was observed, the aspartic acid consumption rate of K. catenaforma was markedly higher than that of other organisms. The consumption of glutamic acid by K. catenaforma was not detected at 24 hr of culture. The difference between the consumption of aspartic acid and glutamic acid in this strain might result from a difference in permeability to the amino acids. We considered that L-glutamic acid might substitute for L-aspartic acid if the uptake of glutamic acid could be increased. A number of detergents were screened for their effect on consumption of glutamic acid. Cetyltrimethylammonium bromide, sodium laurylphosphate, and polyoxyethylene sorbitan monolaurate were found to increase the transport rate of glutamic acid, but not of aspartic acid. A method of producing L-proline from glutamic acid was established with the aid of detergents.     

一种简单实用的谷氨酸浓度测定法

谷氨酸和茚三酮可以发生特有的显色反应 ,在 pH 5～ 6时产物蓝紫色最明显。其颜色深浅与谷氨酸浓度大小有关。用分光光度计可以在其最大光吸收波长处测定其OD值。分别以OD值和相应的一系列浓度的L谷氨酸纯品浓度值为纵坐标和横坐标 ,绘制出 1条标准曲线。谷氨酸发酵液中的谷氨酸含量值即可通过分光光度计测得它与茚三酮在最适 pH处反应得到的蓝紫色产物的OD值 ,然后从上述标准曲线上查到。误差率在 1 0 %左右。这为谷氨酸定量测定提供了一种简单实用的方法。     

产L-谷氨酸工程菌株的诱变选育及其发酵效率

以高产L-谷氨酸的谷氨酸棒杆菌GY1为研究对象,采用ARTP进行全局诱变,进一步提高L-谷氨酸的发酵水平。首先,对谷氨酸棒杆菌GY1原生质体的制备及再生条件进行优化,接着,根据致死率选择最佳的ARTP处理时间,然后,采用96微孔板及摇瓶发酵的方式对突变株进行筛选,最后,对获得的优良突变株进行50 L罐发酵验证。结果显示,溶菌酶浓度为10.0 mg/mL,酶解90 min,原生质体形成率和再生率达到最佳。ARTP最佳处理时间为40 s,致死率达到89.6%,经过初筛与摇瓶复筛,获得突变株YAG117,其摇瓶发酵L-谷氨酸含量达16.3 g/L,较出发菌株提高13.9%,且连续传代五代遗传稳定。50 L补料分批发酵条件下,L-谷氨酸产量在36 h最高,达到216.6 g/L,较出发菌株提高12.9%,糖酸转化率达68.87%,比出发菌株提高了10.2%。ARTP处理GY1菌株原生质体,能够有效积累有利突变,提高突变株发酵生产L-谷氨酸的能力,获得的突变株YAG117也显示了较好的工业化应用潜力。     

Extracellular Accumulation of DNA by Hydrocarbon-utilizing Bacteria

Some hydrocarbon-utilizing bacteria grown on n-paraffin as the sole source of carbon accumulated extracellularly a considerable amount of DNA (0.1 g to 0.6 g per liter) which was free from intact cells and slime materials. This was particularly noted when the strains belonging to Pseudomonad were employed. n-Paraffin was a preferable carbon source for the accumulation of DNA by Pseudomonad, while a strain of Arthrobacter accumulated DNA by growing it on glucose.

The DNA thus accumulated was easily isolated and purified free from other cellular components. The purified DNA was highly polymerized (above 4 × l0⁶ daltons), having the same base composition as the cellular DNA.     

Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants.

Corynebacterium glutamicum is used for the industrial production of glutamate. Excretion of the amino acid may be induced by various means. We have analyzed the characteristics of glutamate excretion induced by two amine surfactants, dodecylammonium acetate (DA) and dodecyltrimethylammonium bromide (DTA). Addition of these surfactants induced an immediate efflux of internal glutamate. It also induced a perturbation of the energetic parameters of the cell (decrease of delta mu H, decrease of the internal ATP concentration). The efflux was not the result of these perturbations: glutamate is taken up by the cells via an ATP-dependent unidirectional active transport system and no efflux took place as a consequence of an artificial decrease of the energetic parameters. In addition, amine surfactants also induced an excretion of other species, in particular potassium. We have tested the possibility that the effluxes result from a permeabilization of the lipid bilayer by analyzing the interactions between the surfactants and liposomes.     

Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.     

Extracellular Accumulation of Organic Acids by Isopropanol-utilizing Microorganisms

Isopropanol-utilizing microorganisms were newly isolated from soils and several of them accumulated two acids in the culture broth, α-ketoglutaric acid being a major one and succinic acid a minor one. Two strains (N–79 and S–1), classified as the genus Mycobacterium, were examined for the cultural conditions with respect to the accumulation of the acids. The accumulation of α-ketoglutaric acid depended greatly on the pH value in the broth, which is required to be kept at around 4 for the maximum accumulation. By means of the pH-controlled culture (at 3.5) with a jar fermentor, strain N–79 accumulated α-ketoglutaric acid at a rate of 0.015 g/liter/hr. The data obtained in this work indicate that the metabolism of isopropanol by strain N–79 probably proceeds via the acetone pathway without the inter-conversion between isopropanol and n-propanol.     

Accumulation of Extracellular Amino Acids by Euglena gracilis

SYNOPSIS. Normal Euglena gracilis , strain z, growing in the light in defined medium (initial nitrogen concentration 140 μ/ml) depletes the medium of all ninhydrin-positive N by the time a cell density of 2 million per ml is reached. A further 2- to 3-fold increase in the cell number takes place in the absence of exogenous N. The N content of an early log phase cell is about 100 picograms but decreases very rapidly as the culture continues to grow, reaching 22 picograms in the stationary phase. When grown in the dark, normal cells take up N somewhat more slowly but the supernatant fluid from saturation cultures is again devoid of N.
At modest cell densities, the permanently bleached strains examined contain less N per cell than do normal strains. The cultures of the bleached strains achieve a maximum density of about 1 to 2 million per ml rather than the 4 to 5 million reached by the normal strain. As a result, supernates from stationary phase cultures of bleached cells still contain a large proportion of the total N supplied.
Paper chromatographic analysis of these supernates reveals several ninhydrin-positive compounds. Most of these have been identified as common amino acids. Some of the properties of two unidentified, ninhydrin-positive compounds are described.