CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP. Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor. The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation. CodY proved to bind GTP in vitro. Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells. Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions. Thus, B. subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation. Our understanding of the relationship between environmental signals and global changes in gene expression is limited by the difficulty in identifying intracellular signaling molecules that interact with key regulatory proteins. This gap is particularly apparent for cases of general nutrient limitation. When Bacillus subtilis cells encounter nutrient limitation and enter stationary phase, a variety of adaptive processes-such as genetic competence, secretion of macromolecule-degrading enzymes, import of secondary nutrients, activation of metabolic pathways, chemotaxis and motility, production of antibiotics, and sporulation-are initiated (Sonenshein 1989). A network of global regulatory proteins modulates the cell's response and regulates the choice between adaptation to poor growth conditions and sporulation (Sonenshein 1989(Sonenshein , 2000Burkholder and Grossman 2000), but the specific signals to which these regulators respond have remained a mystery.Many B. subtilis genes that are expressed early in stationary phase are repressed by CodY (Table 1). Preliminary results indicate that CodY also contributes to regulation of at least two genes (citB, spo0A) whose products are necessary for sporulation (M. Ratnayake- Lecamwasam and A.L. Sonenshein, unpubl. Bolotin et al. 1999).CodY was first identified as a repressor of the B. subtilis dipeptide transport (dpp) operon and was found to be active when cells are grown with an excess of glucose or Casamino acids (CAA) as reported by Slack et al. (1995). During vegetative growth, the dpp operon is also directly repressed by AbrB, a second global regulator of earlystationary-phase genes Strauch 1993;Serror and Sonenshein 1996b), but the repressive effects of nutrient excess are mediat...

Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover.

33Activation of systemic acquired resistance in plants is associated with transcriptome 34 reprogramming induced by the unstable coactivator NPR1. ubiquitination and proteasomal degradation of NPR1 are thought to facilitate 36 continuous delivery of active NPR1 to target promoters, thereby maximising gene 37 expression. Because of this potentially costly sacrificial process, we investigated if 38 ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. 39Here we show ubiquitination of NPR1 is a processive event in which initial modification 40 by a Cullin-RING E3 ligase promotes its chromatin association and expression of target 41 genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it 42 is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are 43 opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 44 longevity. Thus, immune-induced transcriptome reprogramming requires sequential 45 actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune 46 activity of NPR1 without strict requirement for its sacrificial turnover. 47 48 Keywords: NPR1, salicylic acid, systemic acquired resistance, plant immunity, 49 ubiquitin. 50Upon activation of SAR, NPR1 is subject to an array of post-translational 76 modifications. A combination of alterations in redox-based modifications, 77 phosphorylation and SUMOylation of NPR1 result in the formation of a transactivation 78 complex that induces the transcription of immune-responsive target genes (Skelly et 79 al., 2016;Withers and Dong, 2016). Subsequent to these post-translational control 80 points, NPR1 becomes phosphorylated at Ser11 and Ser15, which surprisingly results 81 in recruitment of CRL3 followed by its degradation (Spoel et al., 2009). 82Pharmacological inhibition of the proteasome, genetic mutation of CRL3, and mutation 83 of Ser11/15 all stabilised NPR1 protein, yet impaired the expression of SA-induced 84 NPR1 target genes (Spoel et al., 2009). These findings indicate that paradoxically, 85 ubiquitination and degradation of NPR1 are required for the full expression of its target 86 genes. We previously proposed a proteolysis-coupled transcription model in which 87 activation of target gene transcription results in NPR1 being marked as 'spent' by 88 Ser11/15 phosphorylation (Spoel et al., 2009). SUMOylation of NPR1 was required for 89 Ser11/15 phosphorylation and facilitates its interaction with other transcriptional 90 activators (Saleh et al., 2015), suggesting that NPR1 becomes inactivated only after it 91 has initiated gene transcription. Removal of inactive NPR1 from target promoters may 92 be necessary to allow binding of new active NPR1 protein that can reinitiate 93 transcription, thereby correlating the rate of NPR1 turnover to the level of target gene 94 expression (Spoel et al., 2009). This type of transcriptional control by unstable 95 (co)activators has also been reported in other eukaryotes, including for key 96 transcrip...

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