The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of β-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, andcdg3. Within each cdg operon, apdeA gene lies upstream of a dgc gene.cdg1 contains two additional flanking genes,cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1contributes 80% of cellular DGC and PDEA activities andcdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

A protein which specifically binds cyclic diguanylic acid (c-di-GMP), the reversible allosteric activator of the membrane-bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c-di-GMP binding is of high affinity (ÄD 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K + . The c-di-GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c-di-GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.