Determination of the growth rate-regulated steps in expression of the Escherichia coli K-12 gnd gene.
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.
Pease
A J
AJ
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
Wolf
R E
RE
D000085:Acetates / Q000494:pharmacology; D002455:Cell Division; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D005947:Glucose / Q000494:pharmacology; D006207:Half-Life; D008954:Models, Biological; D010734:Phosphogluconate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D000085:Acetates; D012333:RNA, Messenger; D010734:Phosphogluconate Dehydrogenase; D005947:Glucose