Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

Abstract

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285°on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3-to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

Structural and Functional Basis for (S)-Allantoin Formation in the Ureide Pathway

“…No residues are in the disallowed region defined by the program PROCHECK (23). The figures were prepared using PyMOL, 4 and the structure was analyzed using the CCP4 suite (23).…”

“…Subsequently, (S)-allantoin, which contains an even ratio of nitrogen to carbon, is used as a nitrogen source through further enzyme-dependent degradation. This metabolism of uric acid, a process named the ureide pathway, has a pivotal role in transforming the nitrogen that is fixed in leguminous plants (2,3) and also plays a crucial role in some bacteria under nitrogen-limited conditions (4). This pathway was once thought to be executed by a single enzyme, urate oxidase (5), but recent investigations have revealed two additional enzymes in the pathway (Scheme 1).…”

Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family

“…To date, a total of 49 genes belonging to the TRP family have been identified from diverse species, including bacteria, plants, and animals (12). In some cases TRP genes are physically located in the ureide pathway-related gene clusters (14). Despite the high degree of sequence similarity between TRP and TTR, distinct differences appear in the sequences, particularly at the …”

“…To date, a total of 49 genes belonging to the TRP family have been identified from diverse species, including bacteria, plants, and animals (12). In some cases TRP genes are physically located in the ureide pathway-related gene clusters (14). Despite the high degree of sequence similarity between TRP and TTR, distinct differences appear in the sequences, particularly at the C terminus, although the sequence Tyr-Arg-Gly-Ser is strictly conserved in all members of the TRP family ( Here, we present the crystal structures of PucM and its complexes with the slow substrate 5,6-diaminouracil (DAU) (9) and the urate analog 8-azaxanthine (AZX) (15,16) to investigate the proposed functional role of PucM as HIUHase.…”

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052

“…PucR regulators are responsible for regulating purine catabolism (Schultz et al, 2001). In SA-1, the SNV changed a histidine residue, positively charged, for a polar uncharged asparagine residue.…”