Department of Pharmaceutical Biotechnology, Pharmaceutical Sciences Research Center, School of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
Uricase or Urate oxidase (urate:oxygen oxidoreductase, EC 22.214.171.124), a peroxisomal enzyme which is found in many bacteria, catalyzes the oxidative opening of the purine ring of urate to yield allantoin, carbon dioxide, and hydrogen peroxide. In this study, the phylogeny of urate oxidase (uricase) producing bacteria was studied based on gene sequences of 16S rRNA and uricase protein. Representative and type strains (52 strains total) of most of the known species were analyzed. The acquired sequences (rDNA sequences of the 16S rRNA genes and the amino acid sequences of uricase) were aligned with the Clustal W program using MEGA software version 4.0. Phylogenetic trees were constructed with the neighbor-joining method, and were bootstrapped with 500 replications of each sequence. The large congruence of phylogenetic relationship between the uricase gene and of 16S rRNA gives considerable support to the phylogeny of urate oxidase producing bacteria which was previously suggested on the basis of 16S rRNA sequences. The observed consistency promotes the idea that each of these genes shared a common evolutionary history in uricase producing bacteria we have analyzed.
Uricase or Urate oxidase (urate:oxygen oxidoreductase, EC 126.96.36.199), a peroxisomal enzyme, catalyzes the oxidative opening of the purine ring of urate to yield allantoin, carbon dioxide, and hydrogen peroxide [1-2]. The enzyme is a tetramer, consisting of two types of different subunits with a final molecular weight ranging from 145 to 150 kDa . A number of bacteria are able to produce uricase, including, but not limited to, Pseudomonasaeruginosa, Arthrobacter globiformis, Bacillus subtilis, Bacillus fastidious, Nocardia farcinica and Microbacterium sp. [4-12]. This enzyme has also been reported in fungi, plants and animals [13-15]. It has a unique evolutionary feature, in that the enzyme has been lost during primate evolution with no obvious explanation . The biological reason for the loss of urate oxidase activity in humans and certain primates is unknown. According to one view, this loss has had a distinctly beneficial effect.
It has been shown that uric acid is a powerful antioxidant and a scavenger of free radicals; therefore, a high serum uric acid level caused by the loss of urate oxidase activity may have contributed to a decreased cancer rate and a lengthened hominoid life span . In order to investigate the genetic relationships among the uricase producing bacteria further, sequences of another gene not related to the 16S rRNA should be considered. Therefore, in this report, we compare the genetic relationships of the uricase producing bacteria based on 16S rDNA sequences and on uricase amino acid sequences.
2. Material and Methods
Here, representative and type strains (52 strains total) of most of the known bacterial species were analyzed. Almost all 16S rDNA sequences and uricase amino acid sequences were collected from the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov). List of all these 52 strains and the GenBank accession numbers for genomes used in this study are listed in Table 1.
The acquired sequences (rDNA sequences of the 16S rRNA genes and the amino acid sequences of uricase) were aligned with the Clustal W program using MEGA software version 4.0 . Phylogenetic trees were constructed with the neighbor-joining method, and were bootstrapped with 500 replications of each sequence.
Figure 1. Neighbor-joining tree constructed from amino acid sequences of the uricase showing the phylogenetic relationships. Bootstrap values above 50 are indicated at the main nodes. Bootstrap values are given above branches.
Figure 2. Neighbor-joining tree constructed from nucleotide sequences of the 16S rDNA gene showing the phylogenetic relationships. Bootstrap values above 50 are indicated at the main nodes. Bootstrap values are given above branches.
Table 1. Uricase producing bacteria included in this study.
GenBank Accession number
bv. viciae 3841
So ce 56
Phylogeny according to 16S rDNA sequences and uricase amino acid sequences
The phylogenetic tree based on uricase amino acid sequences (Figure 1) revealed a tree topology which was generally similar to the 16S rDNA tree (Figure 2). The large congruence of phylogenetic relationship between the uricase gene and of 16S rDNA gives considerable support to the phylogeny of urate oxidase producing bacteria previously suggested on the basis of 16S rDNA sequences. The similar position of
Strepomyces, Bacillus, Arthrobacter andErwinia species indicate that the phylogenetictree based on uricase amino acid sequences (Figure 1) was highly consistent with the 16S rDNA tree (Figure 2), suggesting that each of these genes shared a common evolutionary history in uricase producing bacteria we have analyzed in this study.
The evolutionary history of tree constructed from nucleotide sequences of the 16S rDNA gene was inferred using the Neighbor-Joining method . The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed . Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches . The evolutionary distances were computed using the Maximum Composite Likelihood method  and are in the units of the number of base substitutions per site. The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 971 positions in the final dataset. On the other hand, The evolutionary distances for amino acid based tree were computed using the Poisson correction method  and are in the units of the number of amino acid substitutions per site. The analysis involved 52 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 142 positions in the final dataset.
The infrageneric groups retained in 16S rDNA nucleotide analysis only partly reflect the current taxonomical classification of these bacteria. It should be kept in mind that a more complete sample of urate oxidase species will considerably enhance the phylogenetic resolution and closely reflects the results of analysis.
The uricase-producing bacteria represent a phylogenetically coherent group of bacteria, which are also closely related according to the 16S rDNA sequence. By comparison of the sequences of both genes, an improved relationship for the uricase-producing bacteria could be established. This observation is quite remarkable and strongly supports the 16S-rDNA-based phylogeny of these bacteria. High bootstrap values at most of the branching points suggest that the analysis has a high degree of reliability. However, more detailed studies including a higher number of accessions are necessary to test this hypothesis.
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