Stephen M. Boyle

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

Negative control of orinithine decarboxylase and arginine decarboxylase by adenosine-3′:5′-Cyclic monophosphate in Escherichia coli

SummaryThe polyamine biosynthetic enzymes, ornithine decarboxylase (EC 4.1.1.17) (ODC) and arginine decarboxylase (EC 4.1.1.19) (ADC), are negatively controlled by cAMP in Escherichia coli. The specific activities of ODC and ADC were determined in crude extracts prepared from E. coli strains carrying a mutation in the adenylate cyclase (EC 4.6.1.1) structural gene (cya) and wildtype strains. These strains were cultured on various carbon sources in the presence and absence of cAMP. In wild-type strains, ODC and ADC activities were diminished in cells grown on glycerol compared to these strains cultured on glucose. When cya strains were grown on glucose or glycerol, ODC and ADC activities were the same. Addition of 1 mM cAMP to glucose-based medium repressed ODC and ADC activities in both the wild-type and cya strains. Furthermore, cAMP exerts its negative control through the cAMP receptor protein, since strains carrying a mutation in the crp structural gene fail to repress ODC and ADC activities in response to increased cAMP obtained by carbon source manipulation or cAMP supplementation of the growth medium. This evidence suggests that negative control of ODC and ADC by cAMP occurs at the level of transcription.

Transcription of the speC (ornithine decarboxylase) gene of Escherichia coli is repressed by cyclic AMP and its receptor protein

The speC gene encoding ornithine decarboxylase (ODC) in Escherichia coli is negatively regulated by cAMP and the cAMP receptor protein (CRP). In minicells transformed with the plasmid pODC bearing speC, cAMP supplementation repressed ODC synthesis. In a cell-free protein synthesizing system directed by pODC, cAMP at 10(-5) M repressed ODC synthesis by 90%. This repression required a functional CRP as cAMP failed to repress ODC synthesis in vitro in an extract prepared from a crp- strain; the addition of purified CRP to the crp- extract restored the ability of cAMP to repress ODC synthesis. In a prototroph transformed with the plasmid pCOD bearing a speC::tet chimeric gene, cAMP supplementation decreased tetracycline (Tc) resistance. In contrast, in crp- strains transformed with pCOD or pTET (TcR), cAMP supplementation did not change their Tc resistance. When a cya- strain was supplemented with 2 mM cAMP, steady state levels of ODC mRNA were repressed by 80%. However, when a isogenic crp- strain was supplemented with 2 mM cAMP, no repression of ODC mRNA was observed. These results indicate that the cAMP-CRP complex exerts negative transcriptional control of ODC synthesis as a function of the speC promoter.

Polyamine levels in Escherichia coli during nutritional shiftup and exponential growth

At different exponential growth rates obtained either by varying the carbon source of the culture medium or limiting glucose uptake, intracellular levels of putrescine and spermidine were measured. Over a ten-fold increase in growth rate an approximately three-fold increase in putrescine level and a 3.5-fold increase in spermidine level per cell absorbance were observed. Conditions favoring an abrupt alteration in growth rate, such as occur following nutritional shiftup of Escherichia coli, resulted in a significant increase in the intracellular level of putrescine and virtually no change in the spermidine level. Because of the magnitude and the timing of the change in polyamine levels, the hypothesis that polyamines are (the components) responsible for inducing the rapid increase in the rate of RNA synthesis following nutritional shiftup is rejected.

The relationships among RNA synthesis, RNA polymerase synthesis and guanosine tetraphosphate levels in Escherichia coli during nutritional shift-up

Abstract The relationships among the rate of RNA synthesis, RNA polymerase synthesis and activity, and guanosine tetraphosphate levels were investigated following nutritional shift-up in Escherichia coli. RNA synthesis continues at the preshift rate for 1.5 min after which an increase is observed that reaches a new steady-state rate at between 2 and 2.5 min. RNA polymerase activity measured in crude extracts increases immediately and by 10 min has increased 50%. RNA polymerase synthesis as measured by the synthesis of the β and β′ subunits lags for 2.5 min and then increases 75% by 10 min. Guanosine tetraphosphate levels decrease 50% by 3 min to levels characteristic of steady-state post-shift-up cells. The significance of these data to the regulation of RNA synthesis during shift-up is discussed.

Activity of polypeptide chain initiation factors during a nutritional shift-up in Escherichiacoli

Summary During the first 10–15 minutes following nutritional shift-up in Escherichia coli , no significant increase was detected in the specific activity of initiation factors. The next 25 minutes, however, were characterized by a doubling of specific activity of these factors. Measurements of initiation factor activity of different exponential growth rates revealed a linear proportionality between growth rate and initiation factor activity. For each doubling of growth rate, initiation factor activity increased 1.6–1.8 fold.

Protein synthesis during a nutritional shift-up in Escherichia coli

Abstract The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in Escherichia coli ; while the rate of synthesis of elongation factors did not increase until 5–10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron.

Synthesis of messenger RNA coding for elongation factors G and Ts during nutritional shift-up in Escherichiacoli K-12

Abstract Following a nutritional shift-up, the rate of ribosomal protein synthesis increased almost immediately, whereas the rates of synthesis of elongation factors G and Ts increased after a lag of 5 – 10 minutes. The messenger RNA content during shift-up was examined using a technique involving labelling of the protein synthesized following inhibition of RNA synthesis. Messenger coding for the ribosomal proteins increased immediately, whereas that coding for the elongation factors G and Ts increased only after a lag of about 10 minutes and closely followed the pattern of messenger coding for total soluble proteins. These data suggest that the observed differences in rates of synthesis of the ribosomal and elongation factor proteins during shift-up are due to differences in the tiems of transcription of the messengers. The possibility of translational discrimination is discussed.