Nucleotide sequence of arfB of Clostridium stercorarium, and prediction of catalytic residues of alpha-L-arabinofuranosidases based on local similarity with several families of glycosyl hydrolases.

The nucleotide sequence of the alpha-L-arabinofuranosidase gene arfB from Clostridium stercorarium was determined. The deduced protein has a molecular mass of 56.2 kDa with an amino terminus identical to the N-terminal sequence of the purified mature enzyme from C. stercorarium. Its sequence is homologous to arabinofuranosidases of glycosyl hydrolase family 51. Sequence alignment and cluster analysis reveal three new members of glycosyl hydrolase family 51, allowing for the definition of highly conserved regions. Two of these regions are remarkably similar to the most conserved regions within several other families of glycosyl hydrolases, which have in common a (beta/alpha)8-barrel as the core super-secondary structure, and allow to predict the acid/base catalyst and the nucleophile of the active site.

Bacterial phylogeny based on comparative sequence analysis.

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Phylogenetic relationships of Bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes.

Comparative sequence analyses were performed on 14 genes encoding bacterial elongation factors EF-Tu and 7 genes encoding the beta-subunit of bacterial F1F0 type ATP-synthases. The corresponding predicted amino acid sequences were compared with published primary structures of homologous molecules. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. The EF-Tu data were in very good agreement with the rRNA data, although the resolution within the EF-Tu tree was reduced at certain phylogenetic levels. The resolution power of the ATPase beta-subunit sequence data were more reduced than those of the EF-Tu data. In comparison with the 16S rRNA tree there are minor differences in the order of adjacent branchings within the ATPase beta-subunit tree.