Glycerol auxotrophs

Nakao, Y., Kikuchi, M., Suzuki, M., and Doi, M. (1972) Microbial production of L-glutamic acid by glycerol auxotrophs and production of L-glutamic acid from n-paraffrns. Agric. Biol. Chem., 36, 490-496. 

Studies on Bacterial Membrane Biogenesis Using Glycerol Auxotrophs... 

When glycerol auxotrophs are deprived of glycerol, net phospholipid synthesis stops immediately (Fig. 1). This was found with all glycerol auxotrophs tested. However, incorporation of [ P]-labeled phosphate into lipid continues after glycerol deprivation, albeit at a reduced rate. This in-... 

Fig. 1. Phospholipid synthesis after glycerol deprival. B. subtilis glycerol auxotroph B42 was grown for 3 generations in Tris medium with 20 Mg glycerol/ml and 0.5 mM phosphate and 0.5 Ci [ P]phos-phate/ml, filtered, washed and resuspended in the same medium at the same specific activity of [32p] 20 Mg glycerol/ml (0),...

Fig. 3. Effects of glycerol deprivation on RNA, DNA, and protein synthesis. Exponentially growing cells of B. subtilis glycerol auxotroph B67 were filtered, washed, and resuspended in medium with (O, , A) and without ( , , A) glycerol. At times indicated, samples were removed and RNA (O, ), DNA ( , ), and protein (A, A) were determined. The resulting values were normalized to facilitate comparison. ...

A culture of B. subtilis glycerol auxotroph strain B42 was filtered, washed and resuspended in medium, one-third was harvested by centrifugation, half the remainder was incubated with 20 jxg glycerol/ml, and the other half was incubated without glycerol for 60 min. Membranes were prepared, and protein and lipid phosphorus were determined. The values for 168.2 are shown to enable comparison with a wild-type culture. 

B. subtilis glycerol auxotroph B42 his, trp, glyc, glpD) was grown in medium supplemented with glycerol, histidine, tryptophan, methionine, proline, threonine, serine, and valine, filtered at time 0, and incubated with or without histidine and tryptophan for 80 min. Membranes were isolated, and their composition was determined. 

Fig. 7. Effect of glycerol deprivation on the appearance of permease and / -galac-tosidase activities. A culture of S. aureus glycerol auxotroph S8 grown at 34°C was filtered, washed, and resuspended in medium containing 10 M Gal-6-P at 44°C. Aliquots were removed after 0, 20, 40, 60, and 80 min and assayed for / -galactosi-dase and for uptake of [ CjlPTG. Cells were incubated in the presence (O) and absence ( ) of glycerol. ...

Several possibilities exist to explain or rationalize the cessation of growth after a doubling in mass. However, the most likely seem to be either a loss of efficiency of energy production from oxidative phosphorylation due to the decrease in the lipid contents of the membrane, or perhaps most likely, the reduction of the cell s ability to concentrate particular molecules essential for biosynthesis or energy production because of defects in the permeation systems themselves, or increased leakiness of the membrane. In the case of glycerol auxotrophs of S. aureus, it has been found that the specific activity of glycine transport diminishes with time, indicating either a lack of new synthesis of the permeation system or a loss in its efficiency. ... 

Cells can divide in the absence of phospholipid synthesis. All the glycerol auxotrophs examined show one slow division after the cessation of phospholipid synthesis. The only case where division does not occur is in the case of germinating spores of glycerol auxotrophs of B. subtilis. In this case, it was found that DNA synthesis stopped somewhere around the middle of the replication cycle. Since a relationship between the termination of a round of DNA chromosome replication and the initiation of division has been demonstrated, the lack of division in the germinating spores of B. subtilis in the absence of lipid synthesis is understandable. 

Fig. 11. Effect of glycerol on bacteriochlorophyll synthesis. A semianaerobically growing culture of R. capsulata glycerol auxotroph C2 was filtered, washed, and resuspended with (O) or without glycerol ( ). Samples were removed for bacteriochlorophyll determinations at various times. After 135 min, glycerol was added back to half of the deprived culture (A) (N. Klein, thesis).

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