Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4- to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10(-5)m and 6.7 x 10(-5)m, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.     

Mutagenesis of Clostridium butyricum

Mutagenesis of Clostridium butyricum ATCC 19398 was best accomplished by u. v. irradiation (254 nm) and methyl methane sulphonate. Nitrosoguanidine was only slightly mutagenic for this organism. Procedures are described which yielded a variety of auxotrophic and antibiotic-resistant mutant strains.     

Photoreactivating capacity in Clostridium butyricum and Clostridium acetobutylicum

No difference in survival was observed when u.v.-irradiated clostridial cells were subsequently incubated in the dark or exposed to photoreactivating light. This suggests that photoreactivation does not occur in Clostridium butyricum and in Clostridium acetohutylicum.     

Superoxide dismutase in Clostridium butyricum spores

Superoxide dismutase was found for the first time in the spores of the anaerobic bacterium Clostridium butyricum. The prosthetic group of the enzyme was shown to contain iron. The enzyme was demonstrated to be highly thermostable.     

酪酸梭菌活菌散在治疗母乳性黄疸中的应用

目的观察在常规治疗的基础上加用酪酸梭菌活菌散（商品名：宝乐安）治疗新生儿母乳性黄疸的疗效。方法176例母乳性黄疸患儿随机分为治疗组和对照组,治疗组90例,对照组86例,对照组采用常规治疗,治疗组在常规治疗基础上加服酪酸梭菌活菌散,2组均不停止母乳喂养,并观察2组患儿日均总胆红素水平及黄疸消退时间的变化。结果治疗组日均胆红素下降值为（58．61±26．52）μmol／L,显著高于对照组（39．12±25．41）μmol／L（P〈0．01）;黄疸消退时间,治疗组为（4．25±2．68）d,显著短于对照组（6．42±2．74）d（P〈0．01）。结论在常规治疗的基础上加用酪酸梭菌活菌散治疗母乳性黄疸,可迅速降低胆红素水平,缩短治疗时间。     

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Biological activity of a polar lipid of Clostridium butyricum spores

A fraction of polar lipids was isolated from spores of the anaerobic bacterium Clostridium butyricum 35/11 exerting a noticeable radioprotective effect. The main biological activity of spore extracts was associated with this fraction. The fraction of polar lipids inhibited autolysis of the bacterial cell walls. The fraction was found to contain a phenolic glycolipid and a peptide component. The bacteriostatic and radiotherapeutic properties of the fraction are presumed to be due to its membranotropic activity.     

Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H₂, it can produce acids (lactic, acetic and butyric acids) and CO₂, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H₂ yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H₂ yield compared with the wild type strain. However, under low partial pressure of H₂, the hbd-deficient strain showed increased H₂ production with the simultaneous decrease of ethanol production, indicating that H₂ production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H₂ production by C. butyricum.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidyltransferase of Clostridium butyricum: specificity for diacylphosphoglycerides

Phosphatidyltransferase from Clostridium butyricum, which catalyzes transfer of the phosphatidyl moiety of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) or phosphatidylserine to primary alcohols such as glycerol, serine and ethanolamine, was tested for its ability to catalyze transfer of the plasmenyl moiety from plasmalogen analogs of PE or PG to glycerol or ethanolamine, respectively. The cell membrane of C. butyricum contains high proportions of these plasmalogens. When diacyl and plasmalogen forms of PE or PG were supplied as donors in equimolar amounts to membrane particles, the diacyl forms were the preferred donors by approx. 7 and 10 to 1, respectively. When the molar ratio of PE its plasmalogen was 1:3.3, the ratio of PG formed to its plasmalogen was 1:0.66. These results show that the enzyme(s) can catalyze transfer of both the diacyl and alkenyl acyl forms of glycerophospholipids, but the diacyl forms are used preferentially.     

产1,3-丙二醇菌株丁酸梭菌的诱变育种

甘油由丁酸梭菌转化成1,3-丙二醇的研究是厌氧条件下进行。为了获得1,3-丙二醇的高产突变株,以丁酸梭菌为出发菌株进行诱变处理。经过硫酸二乙酯（DES）化学诱变得到2株高产正突变株C.but2031和C.but2046,再经过紫外线和亚硝基胍（NTG）复合诱变得到突变株C.but3037。经过初筛、复筛和传代实验,表明其是稳定的突变株。C.but3037的1,3-丙二醇产量由出发菌株的2.2g/L提高到15.7g/L,提高了6.13倍,     

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids

Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)     

酪酸梭菌的研究与应用进展

酪酸梭菌属于微生态制剂,酪酸梭菌具有抑制肠出血性大肠埃希菌、痢疾志贺菌、霍乱沙门菌、霍乱弧菌等肠道致病菌的生长繁殖,抑制艰难梭菌生长繁殖,促进双歧杆菌、乳酸菌等肠道有益菌的生长繁殖,加强肠道黏膜膜营养代谢、保护受损的黏膜屏障,激活免疫细胞、改善并恢复肠道正常免疫力,防治肿瘤的生长的作用.所以酪酸梭菌制剂对腹泻、肠炎、肠易激综合征、结直肠癌新生儿黄疸等疾病都有很强的防治作用.     

Rapid Regulation of an Anthranilate Synthase Aggregate by Hysteresis

The anthranilate synthase aggregate from Bacillus subtilis is composed of two nonidentical subunits, denoted E and X, which are readily associated or dissociated. A complex of subunit E and X can utilize glutamine or ammonia as substrates in the formation of anthranilate. Partially purified subunit E is capable of using only ammonia as the amide donor in the anthranilate synthase reaction. The stability of the EX complex is strongly influenced by glutamine and by the concentrations of the subunits. Glutamine stabilizes the aggregate as a molecular species in which the velocity of the glutamine-reactive anthranilate synthase is a linear function of protein concentration. In the absence of glutamine the aggregate is readily dissociated following dilution of the extract; that is, velocity concaves upward as a function of increasing protein concentration. Reassociation of the EX complex is characterized by a velocity lag (or hysteretic response) before steady-state velocity for the glutamine-reactive anthranilate synthase is reached. We propose that association and dissociation of the anthranilate synthase aggregate may be physiologically significant and provide a control mechanism whereby repression or derepression causes disproportionate losses or gains in activity by virtue of protein-protein interactions between subunits E and X.     

酪酸梭菌活菌散预防新生儿高胆红素血症临床观察

目的观察酪酸梭菌活菌散(商品名:宝乐安)预防新生儿高胆红素血症的临床疗效。方法将产科分娩的正常足月新生儿212例,随机分为2组,预防组106例,对照组106例,2组均在生后半小时内开始喂奶。预防组喂服酪酸梭菌活菌散500 mg/次,3次/d,用至黄疸消退,观察黄疸变化情况及不良反应。结果出生后48 h内胎便转黄率预防组为56.6%,对照组为26.4%;生后7 d内皮肤巩膜黄染达高峰预防组为58.5%,对照组为43.3%;高胆红素血症发生率预防组为7.5%,对照组为22.6%;出生后7 d内血清胆红素达高峰率预防组为62.3%,对照组为47.1%。以上各指标2组间差异均具有统计学意义(P0.01或0.05)。结论酪酸梭菌活菌散预防新生儿高胆红素血症安全有效,值得临床推广应用。