Simple downshift and resulting lack of correlation between ppGpp pool size and ribonucleic acid accumulation

M T Hansen, M L Pato, S Molin, N P Fill, K von Meyenburg
1975 Journal of Bacteriology  
The growth rate of Escherichia coli can be limited by the availability of carbon and energy. To impose such a limitation, a-methylglucoside (aMG), a non-metabolizable analogue, can be used to decrease uptake of glucose by competition for the transport of this sugar. Varying the ratio of glucose to aMG allowed shifts in growth rate without simultaneous qualitative changes in the growth medium and permitted examination of the immediate changes accompanying such shifts. Stringent (rel+) as well as
more » ... relaxed (relV) strains were able to rapidly curtail their accumulation of ribonucleic acid (RNA) after a downshift imposed by decreasing glucose transport into the cell. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) accumulated in both rel+ and relstrains after a decrease in growth rate. However, the accumulation of ppGpp in relaxed derivatives was very slow, and there was no direct or obligatory correlation between the level of ppGpp and the rate of RNA accumulation. This latter conclusion is supported by measurements of ppGpp levels and rates of RNA accumulation after restoration of maximal growth rates by addition of excess glucose. The macromolecular composition of bacterial cells varies as a function of growth rate. In particular, the number of ribosomes is regulated such that at any growth rate it is just adequate to sustain the corresponding rate of protein synthesis (19) . The growth rate can be varied by utilizing different carbon sources or by adding precursors for macromolecular synthesis to the medium. However, the significance of changes in cell composition during shifts between qualitatively different media is difficult to evaluate. This is particularly true for shifts from one carbon source to another and for downshifts from rich to minimal media. In these cases the cell needs to change its pattern of gene expression to be able to utilize the new carbon source or to synthesize a number of precursors. The chemostat has been widely used to distinguish which regulatory changes are exclusively a function of growth rate and which are attributable to variation in the nutritional richness of the medium (21). In the chemostat, rapid sampling, labeling, and shifts between different growth rates are, however, technically very difficult. We have therefore examined a system that allows changes in growth rate without a simultaneous qualitative change in the composition of the medium. The non-metabolizable analogue a-methylglucoside (aMG) is a competitive inhibitor of glucose uptake in Escherichia coli (12) and can be utilized to limit the rate of growth. The principle in this system is thus to limit the rate of entry of carbon source into the cell, in contrast to limiting the rate with which it is supplied to the medium in the chemostat. The mechanism underlying regulation of ribosomal synthesis is at present poorly understood. Cashel and Gallant (5) first proposed the nucleotides guanosine 5'-diphosphate 3'diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) as effectors limiting the rate of synthesis of ribosomes. Results consistent with this hypothesis (6, 17, 26, 27) as well as some apparently inconsistent (2, 13, 18, 20) have been reported. In the experiments reported in this paper we limited the transport of glucose with aMG and examined the pools of ppGpp and the rate of accumulation of stable ribonucleic acid (RNA) during shifts in growth rate. MATERIALS AND METHODS Strain and growth conditions. Strains NF 541 and NF 542 were prepared by co-transduction of the relAl and fuc+ markers into a leu-, pyrB-derivative of the E. coli B strain AS 19 (24). Among the transductants, NF 541 (AS 19, leu-, pyrB-, luc+, 585 on May 7, 2020 by guest
doi:10.1128/jb.122.2.585-591.1975 fatcat:zgw6yuutcneyrhptrkdheeoyn4