Studies on the metabolism of bacterial lipid granules

Abstract

1. Experiments have been conducted with aaporogenous strains of Bacillus cereus and Bacillus megaterlum.

2. Methods end chemically defined media have been developed for the bulk culture of the organisms. Manganese was found to be an essential additive to the growth medium.

3. A major constituent of the "lipid" granules In these species Is a polymer of ß-hydroxybutyric acid (PHB). The metabolism of PHB has been studied under various conditions.

4. PHBB was formed rapidly during growth and In washed suspensions in conditions of carbon and energy excess and dissimilated in the absence of a utilisable substrate. The formation of PHB has not previously been demonstrated In washed suspensions.

5. A high level of PHB In a cell conferred a high rate of endogenous respiration and delayed autolysis.

6 The formation of PHB showed an optimum around pH 7.5 and growth studies confirmed that It is unlikely to be a "neutralisation mechanism".

7. It was concluded from the above (4 - 6) that PHB acted primarily as a reserve of carbon and energy.

8. Washed suspension experiments showed that PHB was dissimilated more rapidly aerobically than anaerobically. More oxygen was absorbed than would have been required for the complete oxidation of the PHB lost aerobically and more acid was formed anaerobically than was accounted for by a simple hydrolysis of the degraded PHB to ß-hydroxybutyric acid.

9. Quantitative studies showed 90% of the theoretical yield of ß-hydroxybutyric acid and acetoacetic acid from the amount of PHB broken down anaerobically. Little ß-hydroxybutyric acid or acetoacetic acid accumulated aerobically and it appeared that almost complete oxidation of the degraded PHB took place.

10. Chromatographic analyses of the products of PHB breakdown confirmed the presence of ß-hydroxybutyric and acetoacetic acids.

11. The organisms formed PHB in the presence of glucoses pyruvate or ß-hydroxybutyrate.

12. Acetate did not induce synthesis of PHB on its own but caused an extensive increase in the amount formed from the above substrates. With a fixed glucose concentration (0.05 M.) and varied acetate concentrations, PHB formation was proportional to the acetate concentration up to 0.05 M.

13. Various other organic substances were tested as substrates for PHB synthesis. No formation of PHB was effected but several of the substances inhibited its breakdown to some extent.

14. Neither a nitrogen nor a magnesium source was a necessary additive for PHB synthesis.

15. PHB formation from glucose and acetate was strongly inhibited by cyanide and dinitrophenol and less strongly by the co-enzyme A antagonist pantoyl-tauryl anisidide.

16. Low concentrations of fluoroacetete stimulated PHB formation from glucose and acetate, the oxygen consumption being slightly inhibited. The ß-hydroxybutyrate analogue 2-hydroxy-1-propane sulphonate caused a slight increase in PHB synthesis without affecting the oxygen uptake.

17. Pure oxygen inhibited the formation of PHB, though synthesis was optimal with 5% oxygen in nitrogen as the gas phase. Neither B. cereus nor N. megaterium formed PHB under nitrogen but the former organism did so under hydrogen. Carbon dioxide did not stimulate PHB formation.

18. While utilising ß-hydroxybutyrate for the synthesis of PHB the organisms converted much of the substrate to acetoacetate.

19. The hypochlorite method was unsuitable for the estimation of PHB in lysates derived from lysozyme treatment of whole cells. Evidence is led that the ether soluble component from hypochlorite isolated granules may be derived, at least partly, from other cell constituents.

20. Synthesis or degradation of PHB in lysozyme prepared cell-free extracts could not be demonstrated.

21. Neither utilisation of ß-hydroxybutyrate nor initiation of oxygen uptake upon its addition could be shown with the extract.

22. A DPN-linked ß-hydroxybutyric dehydrogenase could be demonstrated in carefully nrepared extracts after a lag period which lengthened on storage. Neither CoA, ATP nor KCN enhanced DPN reduction in these circumstances.

23. Pyruvate was decarboxylated anaerobically with the formation of quantitative amounts of acetaldehyde. The addition of DPT accelerated this process but CoA was inactive.

24. Studies using isotopically labelled substrates showed that acetate was certainly incorporated into PHB during synthesis in glucose ± acetate, and both pyruvate and acetate during synthesis in pyruvate acetate.

25. Possible biochemical pathways of synthesis and degradation of PHB are discussed in the light of these experimental findings.