Abstract
BackgroundIn bacteria, the biosynthesis of polyamines is modulated at the level of transcription as well as post-translationally. Antizyme (Az) has long been identified as a non-competitive protein inhibitor of polyamine biosynthesis in E. coli. Az was also revealed to be the product of the atoC gene. AtoC is the response regulator of the AtoS-AtoC two-component system and it functions as the positive transcriptional regulator of the atoDAEB operon genes, encoding enzymes involved in short chain fatty acid metabolism. The antizyme is referred to as AtoC/Az, to indicate its dual function as both a transcriptional and post-translational regulator.ResultsThe roles of polyamines on the transcription of atoS and atoC genes as well as that of atoDAEB(ato) operon were studied. Polyamine-mediated induction was tested both in atoSC positive and negative E. coli backgrounds by using β-galactosidase reporter constructs carrying the appropriate promoters patoDAEB, patoS, patoC. In addition, a selection of synthetic polyamine analogues have been synthesized and tested for their effectiveness in inducing the expression of atoC/Az, the product of which plays a pivotal role in the feedback inhibition of putrescine biosynthesis and the transcriptional regulation of the ato operon. The effects of these compounds were also determined on the ato operon expression. The polyamine analogues were also tested for their effect on the activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis and on the growth of polyamine-deficient E. coli.ConclusionPolyamines, which have been reported to induce the protein levels of AtoC/Az in E. coli, act at the transcriptional level, since they cause activation of the atoC transcription. In addition, a series of polyamine analogues were studied on the transcription of atoC gene and ODC activity.
Highlights
In bacteria, the biosynthesis of polyamines is modulated at the level of transcription as well as post-translationally
Effect of putrescine and spermidine on the transcription of the atoS-atoC two component system genes and the ato operon The ability of the reporter constructs (Fig 1), carrying lacZ fused to either of the promoters of the atoSC two component system (i.e the atoS or the atoC promoter) or to its regulated genes, to respond to polyamines was evaluated in three E. coli strains
The isogenic E. coli strains, BW25113 and BW28878 that either carry the wild-type atoSC (BW25113) [29] or a deletion of the atoSC genomic region (BW28878) [30] and the MA255 strain [9], were transformed with the recombinant plasmids described at Fig. 1
Summary
The biosynthesis of polyamines is modulated at the level of transcription as well as post-translationally. Antizyme (Az) has long been identified as a non-competitive protein inhibitor of polyamine biosynthesis in E. coli. The antizyme is referred to as AtoC/Az, to indicate its dual function as both a transcriptional and post-translational regulator. Polyamines are indispensable cellular components implicated in many physiological functions, such as DNA replication and repair, transcription, protein synthesis and post-translational protein modifications [1]. The post-translational regulation of ODC is mainly mediated by polyamine-inducible non-competitive protein inhibitor(s), termed antizymes [9,10,11,12,13]. The mammalian antizyme has been found to promote the ubiquitin-independent degradation of ODC by the 26S proteasome [14,15], as well as to negatively regulate polyamine transport [16,17]
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