Determination of the growth rate-regulated steps in expression of the Escherichia coli K-12 gnd gene

Abstract

In Escherichia coli K-12 strain W3110, the amount of 6-phosphogluconate dehydrogenase relative to that of total protein, i.e., the specific enzyme activity, increases about threefold during growth in minimal media over the range of growth rates with acetate and glucose as sole carbon sources. Previous work with gnd-lac operon and protein fusion strains indicated that two steps in the expression of the gnd gene are subject to growth rate-dependent control, with at least one step being posttranscriptional. With both Northern (RNA) and slot blot analyses, we found that the amount of gnd mRNA relative to that of total RNA was 2.5-fold higher in cells growing in glucose minimal medium than in cells grown on acetate. Therefore, since the total mRNA fraction of total RNA is essentially independent of the growth rate, the amount of gnd mRNA relative to that of total mRNA increases about 2.5-fold with increasing growth rate. This indicates that most of the growth rate-dependent increase in 6-phosphogluconate dehydrogenase can be accounted for by the growth rate-dependent increase in gnd mRNA level. We measured the decay of gnd mRNA mass in the two growth conditions after blocking transcription initiation with rifampin and found that the stability of gnd mRNA does not change with growth rate. We also used a gnd-lacZ protein fusion to measure the functional mRNA half-life and found that it too is growth rate independent. Thus, the growth rate-dependent increase in the level of gnd mRNA is due to an increase in gnd transcription, and this increase is sufficient to account for the growth rate regulation of the 6-phosphogluconate dehydrogenase level. The dilemma posed by interpretations of the properties of gnd-lac fusion strains and by direct measurement of gnd mRNA level is discussed.

Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs

“…This relationship has been documented in a number of studies in which transcription increased in response to environmental conditions (Meyer & Schottel, 1991;Pease & Wolf, 1994). However, very few mRNA stability studies have been published.…”

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

“…However, LDH synthesis in S. bovis appeared to be enhanced by the increase in growth rate, which is also true for some enzymes in E. coli (Pease and Wolf, Jr., 1994). Several steps may affect gene expression which could be subject to growth control (e.g., the transcriptional, translational, and mRNA stability levels) (Nomura et al, 1980;Pease and Wolf, Jr., 1994;Resnekov et al, 1992). When S. bovis was grown in batch cultures, excess glucose was initially present and the growth rate was dependent upon the availability of glucose.…”

“…Proteins which do not respond to the changes in the cell doubling time must be synthesized at rates that vary in direct proportion to the growth rate of the cell (Pedersen et al, 1978). However, LDH synthesis in S. bovis appeared to be enhanced by the increase in growth rate, which is also true for some enzymes in E. coli (Pease and Wolf, Jr., 1994). Several steps may affect gene expression which could be subject to growth control (e.g., the transcriptional, translational, and mRNA stability levels) (Nomura et al, 1980;Pease and Wolf, Jr., 1994;Resnekov et al, 1992).…”

Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide

“…The specific activities of these enzymes increase in proportion to increased growth rate during steady-state growth on different carbon sources. Studies on the growth rate-dependent regulation of zwf and gnd have shown that at least part of the increase in the specific activities of these enzymes is attributable to an increase in the transcription rates of the respective genes (25,26).Apart from its basic growth rate-dependent regulation, zwf is also a member of at least two other regulons. Thus, unlike gnd, zwf expression is transcriptionally activated by SoxS during episodes of oxidative stress induced by exposure of E. coli to superoxide-generating agents such as paraquat (17,18,39).…”

“…The specific activities of these enzymes increase in proportion to increased growth rate during steady-state growth on different carbon sources. Studies on the growth rate-dependent regulation of zwf and gnd have shown that at least part of the increase in the specific activities of these enzymes is attributable to an increase in the transcription rates of the respective genes (25,26).…”