Anaerobic growth of Bacillus mojavensis and Bacillus subtilis requires deoxyribonucleosides or DNA

Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191(T) and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions. Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth. The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions. The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B. mojavensis JF-2 for the four deoxyribonucleosides. However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth. The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B. mojavensis JF-2 in the presence of hydroxyurea. B. mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source. These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.     

外源glnA基因干扰大肠杆菌的代谢

采用分子生物学的方法构建了含Bacillus subtilis glnA基因的重组菌株Escherichia coli DH5α(pMD19-glnA),用毛细管电泳和核磁共振对重组菌株的转化谷氨酸的产物进行定性鉴定,并进一步通过荧光定量RT-PCR测定谷氨酰胺合成酶基因(glnA)mRNA水平的相对表达量,最后用SDS-聚丙烯酰胺凝胶电泳对蛋白的相对表达情况进行了分析。结果表明重组菌株并没有增加谷氨酰胺的产量,而是明显增加了γ-氨基丁酸(GABA)的产量。实验表明重组菌株中的glnA基因可以正常转录,但是谷氨酰胺合成酶的蛋白表达量并没有增加。这种外源基因干扰大肠杆菌代谢的现象值得进一步研究。     

Glycerol Metabolism in Bacillus subtilis: Gene-Enzyme Relationships

Bacillus subtilis mutants unable to catabolize glycerol (Glp mutants) were isolated and mapped. The location of the mutations on the chromosome was determined by a density transfer technique and confirmed by PBS1 transduction and transformation. The different mutations were ordered relative to each other. Mutations rendering the cells glycerol auxotrophic were also mapped and found not to be linked to the Glp mutations.     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Degradation of guaiacol glyceryl ether (GGE) by Bacillus subtilis

Summary Bacillus subtilis utilized guaiacol glyceryl ether (GGE) as sole carbon source and catabolized it via guaiacol and catechol. Cell free extracts of GGE grown cells contained high levels of catechol 1,2-dioxygenase and cleaved catechol via the ortho pathway. Nutrients such as sugars, organic acids, methanol, nitrogen and phosphate influenced the catabolism of GGE by B. subtilis.     

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Summary: Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.     

Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.     

Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism)

The onset of anaerobiosis in barley root tissue (Hordeum vulgare L. cv Himalaya) results in the following metabolic responses. There are rapid increases in the levels of pyruvate, lactate, and ethanol. Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue. Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate. The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine. Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions. Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis. Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue. The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.     

Insertion mutagenesis in Bacillus subtilis (Marmur)

A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed. Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants. Chromosomal localization was established and the properties of several mutants were studied.     

Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase

The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive. GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway. Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG-encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real effectors of GltC. Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline. In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA.     

Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis.

In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria. Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase. More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate. Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd. The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B. subtilis appears to be different from that in enteric bacteria. Cytosine cannot be used by Ura-requiring mutants as pyrimidine source. dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase. Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase. This Cyd kinase activity has never been reported for B. subtilis.     

柠檬酸钠对枯草杆菌生长代谢及肌苷积累的影响

研究柠檬酸钠对枯草杆菌生长代谢及产苷的影响 ,在基础料中添加浓度为 0 2g L的柠檬酸钠 ,肌苷产量提高 18% ,肌苷对葡萄糖得率增加 38%。通过分析糖代谢途径中关键酶的酶活 ,结果表明添加柠檬酸钠改变了一些关键酶的活力 ,可降低糖酵解途径中 6_磷酸果糖激酶和丙酮酸激酶的活力 ,从而减弱了糖酵解途径的通量。     

Chelation of calcium ions by poly(gamma-glutamic acid) from Bacillus subtilis(chungkookjang)

Many studies have clarified that poly(gamma-glutamic acid) (PGA) increases the solubility of Ca(2+), suggesting that PGA enhances calcium absorption in small intestine. However, there has been no report on the specific interaction between PGA and Ca(2+) in water. We studied the aqueous solution properties of PGA calcium salt (PGA-Ca complex). The chelating ability and binding strength of PGA for Ca(2+) were evaluated. PGA-Ca complex was soluble in water in contrast with the insolubility of poly(acrylic acid) (PAA) calcium salt and the chelating ability of PGA for Ca(2+) was almost the same than that of PAA. The globular conformation of PGA-Ca complex in water was estimated by SEC and viscosity measurements. The chelation of PGA for Ca(2+) was examined by 1H NMR. The present study showing the characteristics of PGA-Ca complex will provide useful information of the calcium absorption by PGA in vivo.     

Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis

A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.     

Sequential binding and sensing of Zn(II) by Bacillus subtilis Zur

Bacillus subtilis Zur (BsZur) represses high-affinity zinc-uptake systems and alternative ribosomal proteins in response to zinc replete conditions. Sequence alignments and structural studies of related Fur family proteins suggest that BsZur may contain three zinc-binding sites (sites 1-3). Mutational analyses confirm the essential structural role of site 1, while mutants affected in sites 2 and 3 retain partial repressor function. Purified BsZur binds a maximum of two Zn(II) per monomer at site 1 and site 2. Site 3 residues are important for dimerization, but do not directly bind Zn(II). Analyses of metal-binding affinities reveals negative cooperativity between the two site 2 binding events in each dimer. DNA-binding studies indicate that BsZur is sequentially activated from an inactive dimer (Zur(2):Zn(2)) to a partially active asymmetric dimer (Zur(2):Zn(3)), and finally to the fully zinc-loaded active form (Zur(2):Zn(4)). BsZur with a C84S mutation in site 2 forms a Zur(2):Zn(3) form with normal metal- and DNA-binding affinities but is impaired in formation of the Zur(2):Zn(4) high affinity DNA-binding state. This mutant retains partial repressor activity in vivo, thereby supporting a model in which stepwise activation by zinc serves to broaden the physiological response to a wider range of metal concentrations.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.