Studies on the Bacterial Formation of Peptide Antibiotic,Colistin

The biosynthetic mechanism of 6-methyloctanoic and isooctanoic acids, which are present in the amide linkage with the α-amino group of the terminal α, γ-diaminobutyric acid residue of colistin A and B, respectively was investigated. From the isotopic experiments using isoleucine-U-¹⁴C, valine-U-¹⁴C and acetic acid-2-¹⁴C, it was concluded that 6-methyloctanoic and isooctanoic acids were derived from isoleucine and valine, respectively.

Amino acids pooled in colistin-producing cells grown in the synthetic medium were abundant in isoleucine, valine and leucine, which were probable precursors of the abovedescribed fatty acid components of colistin and cellular fatty acids. On the other hand, 6-methyloctanoic and isooctanoic acids were not found in the cellular fatty acids, while C-15 and C-16 branched chain fatty acids usually found in Bacillus sp. were abundantly contained in the cells, indifferently of an improved capacity of colistin formation.     

Studies on the Bacterial Formation of a Peptide Antibiotic,Colistin

The biosynthetic pathway of α,γ-diaminobutyric acid, 6 moles of which are involved in the colistin molecule as a main component, was investigated. On the basis of the isotopic results using aspartic acid-U-¹⁴C as a precursor and also the finding of transaminase activity between α-ketog?utaric acid and α,γ-diaminobutyric acid, though in reverse reaction, α,γ-diaminobutyric acid was proved to be synthesized from aspartic acid via aspartyl-phosphate and aspartic β-semialdehyde. α,γ-Diaminobutyric acid did not inhibit asparto-kinase activity of this bacterium, the first enzyme involved in the process of α,γ-diamino-butyric acid synthesis from aspartic acid, while the end product amino acids such as lysine, threonine and methionine showed inhibition for aspartokinase activity.

On the other hand, α,γ-diaminobutyric acid might be rate-limiting factor in colistin formation, because of stimulatory effect of this diamino acid when added to the medium on colistin production. Furthermore, colistin production appeared to be related with the defect of TCA-cycle and further the resultant increase in activities of the key enzymes such as isopropylmalate synthetase, α-acetolactate synthetase and aspartokinase involved in the biosynthetic pathways of valine, leucine and isoleucine, respectively.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

Isolation of bacteria whose growth is dependent on high levels of CO2 and implications of their potential diversity

Although some bacteria require an atmosphere with high CO(2) levels for their growth, CO(2) is not generally supplied to conventional screening cultures. Here, we isolated 84 bacterial strains exhibiting high-CO(2) dependence. Their phylogenetic affiliations imply that high-CO(2) culture has potential as an effective method to isolate unknown microorganisms.     

Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones

In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrP(Sc)) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrP(C)) at levels similar to the parental N2a cells. In contrast, there was a variation in PrP(C) expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrP(C) at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrP(C) than the parental N2a cells. There was no difference in the binding of PrP(Sc) to prion-susceptible and unsusceptible N2a subclones regardless of their PrP(C) expression level, suggesting that the binding of PrP(Sc) to cells is not a major determinant for prion susceptibility. Stable expression of PrP(C) did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrP(C) at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrP(C) and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.