Sortase has been shown to be a protease that catalyzes the cell wall anchoring of surface proteins containing an LPXTG motif in gram-positive bacteria. In this study, we determined the complete nucleotide sequence of the sortase gene (srtA) of Streptococcus mutans and found a surface protein that was linked to the cell wall by the sortase. The results show that srtA gene of S. mutans consisted of 741 bp and encoded for a sortase protein of 246 amino acids with a molecular weight of 27 489. The deduced amino acid sequence of the S. mutans sortase was highly homologous (65-58%) to those of other Streptococcal species. In a S. mutans mutant lacking sortase, two surface proteins of 200 and 75 kDa were released to the culture supernatant. Western blot analysis with specific antiserum showed that the 200 kDa protein was a surface protein antigen designated PAc. These results suggest that the sortase catalyzes anchoring of the antigen PAc to the cell wall.

In order to clarify the role that sortase (SrtA) plays in anchoring dextranase (Dex) to the cell wall of Streptococcus mutans, both Dex- and SrtA- mutants were constructed by insertional inactivation of the respective genes. Western blot analysis with a Dex antiserum showed that in the srtA mutant the Dex was not bound to the cell wall but was secreted into the culture supernatant. In contrast, in the wild type, Dex remained cell-wall-associated. Biological properties of the srtA mutant were examined in dextran fermentation, colony morphology and adherence to a smooth surface. The srtA mutant, as well as the wild type, retained the ability to ferment dextran. However, the colony morphology of the srtA mutant on Todd Hewitt agar containing sucrose was much larger than that of the wild type and showed a ring-like structure. In addition, the srtA mutant was more adhesive to a smooth surface than the wild type when sucrose was present. However, the adhesion of the srtA mutant remarkably decreased by addition of exogenous dextranase. These studies suggest that the SrtA mediates Dex-anchoring to the cell wall in S. mutans, and cell wall-anchored Dex plays a role in controlling both the adhesive properties of extracellular glucan and the ability to utilize extracellular glucan as a nutrient source. In contrast, extracellular Dex is only responsible for degrading extracellular glucan as a nutrient source.

A sortase-deficient mutant of Streptococcus mutans was prepared by insertional inactivation of a sortase gene (srtA). The srtA mutant was defective in cell wall-anchoring of two surface proteins 200 and 75 kDa in size. A previous study has shown that the 200 kDa protein is a surface protein antigen PAc and that the sortase catalyzes cell wall-anchoring of PAc in S. mutans. In this study another surface protein 75 kDa in size was examined by immunologic and physiologic methods. Western blot analysis with a specific antiserum showed that the 75 kDa protein was a surface protein, glucan-binding protein C. The protein was overexpressed under a stress condition including a sublethal concentration of tetracycline. The srtA mutant cells also lost the ability of dextran-dependent aggregation. These results suggest that the S. mutans sortase mediates cell wall-anchoring of the glucan-binding protein C and dextran-dependent aggregation of this organism.

Major parts of amino-acid-coding regions of elongation factor (EF)-1alpha and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1alpha and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1alpha coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1alpha and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1alpha and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1alphas and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1alpha and EF-2 data sets. The EF-1alpha analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia.

An oral parasite Trichomonas tenax ATCC 30207 synthesizes and secretes various proteinases. By gelatin-SDS-PAGE, we found five proteinases bands (30, 37, 46, 51 and 60 kDa) in cell lysate and four bands (37, 45, 52 and 60 kDa) in culture filtrate. The proteinases hydrolyzed acid soluble type I collagen as well as gelatin. The enzymes were suggested to possess typical characteristics of cysteine proteinases based on the patterns of inhibition and activation by various factors. Based on relative efficiencies of synthetic substrates, most of them were most likely cathepsin B-like enzymes.