Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism

Abstract

The xpt and pbuX genes from Bacillus subtilis were cloned, and their nucleotide sequences were determined. The xpt gene encodes a specific xanthine phosphoribosyltransferase, and the pbuX gene encodes a xanthinespecific purine permease. The genes have overlapping coding regions, and Northern (RNA) blot analysis indicated an operon organization. The translation of the second gene, pbuX, was strongly dependent on the translation of the first gene, xpt. Expression of the operon was repressed by purines, and the effector molecules appear to be hypoxanthine and guanine. When hypoxanthine and guanine were added together, a 160-fold repression was observed. The regulation of expression was at the level of transcription, and we propose that a transcription termination-antitermination control mechanism similar to the one suggested for the regulation of the purine biosynthesis operon exists. The expression of the xpt-pbuX operon was reduced when hypoxanthine served as the sole nitrogen source. Under these conditions, the level of the hypoxanthine-and xanthinedegrading enzyme, xanthine dehydrogenase, was induced more than 80-fold. The xanthine dehydrogenase level was completely derepressed in a glnA (glutamine synthetase) genetic background. Although the regulation of the expression of the xpt-pbuX operon was found to be affected by the nitrogen source, it was normal in a glnA mutant strain. This result suggests the existence of different signalling pathways for repression of the transcription of the xpt-pbuX operon and the induction of xanthine dehydrogenase.

Riboswitches: the oldest mechanism for the regulation of gene expression?

“…Riboswitches regulating various purine biosynthesis and transport genes after binding of purines have been observed in a variety of bacteria and studied experimentally in B. subtilis [28,31], again, despite earlier evidence suggesting the involvement of an unknown protein factor [32]. The purine riboswitch (G-box) regulon in B. subtilis includes pur, xpt -pbuX, pbuG, nupG and pbuE (ydhL) operons [31].…”

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

“…In B. subtilis (Fig. 1), adenine, hypoxanthine, xanthine, uric acid, allantoin and urea all serve as nitrogen sources (Christiansen et al, 1997 ;Cruz-Ramos et al, 1997). The synthesis of urease, encoded by the ureABC operon (Wray et al, 1996), is regulated in response to nitrogen availability and is induced when nitrogen is limiting.…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…NMR samples contained 90:10 H 2 O:D 2 O. For CD-measurements, the ligand hypoxanthine (Christiansen et al 1997;Mandal et al 2003) was added in 20-fold excess to the labeled and unlabeled Gsw apt . Due to the low hypoxanthine solubility, the ligand was added together with 2 mM Mg 2?…”