Identification and disruptional analysis of the Streptomyces cinnamonensis msdA gene, encoding methylmalonic acid semialdehyde dehydrogenase

The msdA gene encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is known to be involved in valine catabolism in Streptomyces coelicolor. Using degenerative primers, a homolog of msdA gene was cloned and sequenced from the monensin producer, Streptomyces cinnamonensis. RT-PCR results showed msdA was expressed in a vegetative culture, bump-seed culture and the early stages of oil-based monensin fermentation. However, isotopic labeling of monensin A by [2, 4-¹³C₂]butyrate revealed that this MSDH does not play a role in providing precursors such as methylmalonyl-CoA for the monensin biosynthesis under these fermentation conditions. Using a PCR-targeting method, msdA was disrupted by insertion of an apramycin resistance gene in S. cinnamonensis C730.1. Fermentation results revealed that the resulting ΔmsdA mutant (CXL1.1) produced comparable levels of monensin to that observed for C730.1. This result is consistent with the hypothesis that butyrate metabolism in S. cinnamonensis in the oil-based fermentation is not mediated by msdA, and that methylmalonyl-CoA is probably produced through direct oxidation of the pro-S methyl group of isobutyryl-CoA. The CXL1.1 mutant and C730.1 were both able to grow in minimal medium with valine or butyrate as the sole carbon source, contrasting previous observations for S. coelicolor which demonstrated msdA is required for growth on valine. In conclusion, loss of the S. cinnamonensis msdA neither affects valine catabolism in a minimal medium, nor butyrate metabolism in an oil-based medium, and its role remains an enigma.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells

Summary Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-¹⁴C]butyrate by HeLa cells was simimar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of α-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of α-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered. This work was supported by Grant CA 21534 from the National Institutes of Health, Bethesda, MD.     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

Effect of sodium butyrate on the hepatoma cell cycle: possible use for cell synchronization

Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [3H]thymidine incorporation studies. Evidence is presented that sodium butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization.     

Development and use of genetic system to identify genes required for efficient low-temperature growth of Psychrobacter arcticus 273-4

We describe the development of genetic tools (electroporation, conjugation, vector for targeted gene replacement) for use in the psychrophile Psychrobacter arcticus 273-4 to test hypotheses about cold adaptation. Successful electroporation only occurred with nonstandard parameters, such as: electrocompetent cells freshly prepared from stationary-phase cultures, high field strengths (25 kV cm⁻¹), long recovery times (16–24 h), and selection with low concentrations of antibiotics. Transformation frequencies were greatly affected by a methylation-dependent restriction barrier homologous to DpnI. The vector pJK100 (which was self-transmissible and contained a Pir-dependent R6K origin of replication) proved effective as a suicide plasmid that could be used to recombine mutations into the P. arcticus 273-4 genome. We used this vector for targeted replacement of dctT, the substrate-binding periplasmic subunit of a TRAP (tripartite ATP-independent periplasmic) transporter (which we have named dctTUF), as it was more highly expressed at cold temperatures. The replacement of dctT (with kan) decreased the rate of growth at low temperatures in mineral medium with glutamate, acetate, butyrate, and fumarate, but not with pyruvate suggesting that DctTUF participates in the transport of glutamate, acetate, butyrate, and fumarate at cold temperatures. This is the first report to demonstrate the creation of site-specific mutants in the genus Psychrobacter, their affect on low-temperature growth, and a substrate range for TAXI proteins of TRAP transporters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

丁酸钠和pH值对离体蚕豆花药中小孢子有丝分裂的影响

含单核中后期至后期花粉的蚕豆离体花药,经过2mM丁酸钠24小时预处理后,再漂浮培养于pH5.8或7.0的液体培养基。以不经预处理的作为对照。结果如下: 1.培养后9天内,丁酸钠预处理和培养基pH值并不明显影响花粉退化百分率。2.培养初期,pH7.0显著促进小孢子不等分裂。3.丁酸钠预处理抑制培养初期的小孢子有丝分裂,而后又显著增加小孢子均等分裂百分率。4.丁酸钠预处理导致小孢子有丝分裂类型的趋向改变。本文还对丁酸钠导致有丝分裂类型趋向改变的可能原因,进行了讨论。     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

Effect of sodium butyrate on the hepatoma cell cycle: Possible use for cell synchronization

Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [³H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization. This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche Scientifique (L. T. and J. K.).     

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO₂ or an auxiliary oxidant. NO _- ³ , N₂O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO _- ³ was reduced extensively to NO _- ³ , TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO₂ or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.Abbreviations DMS dimethylsulphide - DMSO dimethylsulphoxide - TMA trimethylamine - TMAO trimethylamine-N-oxide - NMR nuclear magnetic resonance     

Novel Characteristics of Bifidobacterium bifidum in Solid State Fermentation System

Summary The characteristics of Bifidobacterium bifidum grown in solid state fermentation (SSF) system (water content of media 54.5 and 68.8%) was compared with the submerged fermentation (SmF) system (water content of medium: 89.8%). Besides lactic acid (lactate) and acetic acid (acetate), the bacterium was able to secrete propionic acid (propionate) and butyric acid (butyrate) under SSF conditions. However, it only produced lactate and acetate under SmF conditions. The ratio of lactate to acetate was 1.26–1.62:1 in SSF but it was 1:2 in SmF. A higher content of C16:0 and C18:1 as well as a lower content of C18:0 cell membrane fatty acids were observed in SSF than in SmF. There was a lower growth rate, a lower viable count and a longer logarithmic growth phase for B. bifidum cultivated in SSF than in SmF.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K _m of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1. Received: 3 December 1998 / Accepted: 18 February 1999     

一株东方醋酸杆菌分离与其促进果蝇生长发育

【目的】分离与鉴定黑腹果蝇体内醋酸杆菌,并研究其对宿主生长发育的促进作用。【方法】利用醋酸杆菌选择性培养基分离果蝇肠道醋酸杆菌;通过革兰氏染色和16S rRNA基因比对鉴定菌种;肠道定植实验验证共生关系;发育历期和生长速率实验检测其促进果蝇生长作用;免疫荧光染色技术检测肠道细胞增殖;RT-PCR法检测促生长的分子标志物和相关的信号通路。【结果】菌株为东方醋酸杆菌(Acetobacter orientalis),可以持续地定植在果蝇肠道及其培养基中,并且明显促进果蝇的生长。东方醋酸杆菌通过胰岛素信号通路增加肠分裂细胞的数量和促进蜕皮激素的分泌。【结论】东方醋酸杆菌是果蝇的一种共生菌,对果蝇肠道结构和机体发育具有重要的作用。     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Poly-β-hydroxyalkanoate in Syntrophomonas wolfei

The production of poly--hydroxyalkanoate (PHA) in Syntrophomonas wolfei grown in pure culture or in coculture with Methanospirillum hungatei was studied. PHA was produced by S. wolfei during the exponential phase of growth under both of these cultural conditions. S. wolfei in pure culture also produced PHA in stationary phase when the medium was supplemented with high concentrations of the substrate crotonate. In S. wolfei, PHA levels decreased after growth stopped and most of the substrate was depleted. Altering the C to N ratio of the medium did not affect the amount of PHA made per mg of protein. The incorporation of labeled butyrate but not acetate into PHA during the early stages of growth of S. wolfei and the fact that some of the PHA in a pure culture of S. wolfei grown with trans-2-pentenoate was a polymer of 5-carbon monomer units indicated that a pathway exists for the synthesis of PHA without degradation of the substrate to acetyl-CoA. During the later stages of growth, PHA was made by a pathway which was in equilibrium with the acetate pool. These data indicate that PHA served as a carbon/energy reserve material in S. wolfei, but the synthesis of this polymer was regulated in a manner different from the known pathways.