No abstract

The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.

No abstract

We have shown in previous studies that the glucosyltransferase (Gtf) enzymes of Streptococcus mutans have distinct properties when adsorbed to a surface. In the present study, we compared the activity of Gtf from Streptococcus sanguis, designated GtfSs, in solution and on the surface of saliva–coated hydroxyapatite (sHA) beads, and determined the ability of its product glucan to support the adherence of oral microorganisms. Gtf from S. sanguis 804 NCTC 10904 was purified from culture supernatant fluids by means of hydroxyapatite chromatography. Enzyme and the substrate were prepared in buffers at pH values from 3.5 to 7.5. Maximum activity of GtfSs occurred between pH 5.5 and pH 6.5, whether in solution or adsorbed onto a surface. The solubilized and insolubilized enzymes showed highest activity at 40°C; activity was reduced by 50(±2)% at 20 and 30°C. The enzyme did not form glucans in either phase at 10 or 60°C. The K_m, determined from Lineweaver–Burk plots, for the enzyme in solution was 4.3(±0.4) mmol/l sucrose, and the K_m for the enzyme on sHA beads was 5.0(±1.0) mmol/l sucrose. The ability of the GtfSs glucan synthesized on the surface of sHA beads to support the adherence of oral bacteria was investigated. ³H–thymidine–labeled bacteria (S. mutans GS–5, S. sobrinus 6715, S. sobrinus 6716, S. sanguis 10904, Actinomyces viscosus OMZ105E, A. viscosus 2085, and A. viscosus 2086) were incubated with sHA beads coated with GtfSs glucan. S. mutans GS–5 displayed the highest level of binding numerically. These results show that the GtfSs of S. sanguis is active on sHA beads, that the pH optimum for activity on a surface differs slightly from that in solution, and that its product glucan can support the adherence of oral microorganisms.

OBJECTIVE: Previous studies have shown that glucosyl‐transferase enzymes (Gtfs) of Streptococcus mutans adsorbed to saliva coated hydroxyapatite (sHA) have distinct properties from the same enzymes in solution. The purpose of the present study was to determine the effects on enzyme activity of polyclonal antibodies raised to Gtfs in a soluble form and bound to sHA. MATERIALS AND METHODS: Antiserum was raised in six New Zealand White rabbits using the purified glucosyl‐transferase enzymes (Gtfs) of S. mutans, GtfB, GtfC, or GtfD as soluble antigens or adsorbed to hydroxyapatite (HA, insolubilized). The antisera were examined for their ability to react to these Gtfs and Gtf from Streptococcus sanguis (GtfSs) in Western blot formats as well as inhibit enzyme activity in solution and insolubilized. RESULTS: Antibodies raised against GtfB or GtfC detected all Gtf enzymes examined in Western blots; antibodies raised to GtfD reacted strongly to GtfD and GtfSs, poorly to GtfC, and was non‐reactive with GtfB. Antibodies to GtfB or GtfC inhibited activity of GtfB and GtfC in solution by 90% or more. Enzyme activity adsorbed to sHA was inhibited from 70% to 80% by the same antisera. These same antibodies possessed no specific effect on the activities of either GtfD or GtfSs. Antibodies raised to the GtfD enzyme inhibited activity of GtfD (80% to 90% inhibition) and GtfSs activity (50% to 80%) in solution. In contrast the GtfD antibodies had no effect on the activity of either GtfB or GtfC enzymes in solution. Modest inhibitory effects were noted on GtfC and GtfSs enzymes bound to sHA, but no inhibition was observed for sHA‐bound GtfB or GtfD. CONCLUSION: These data show that some antibodies effective against enzymes in solution may have significantly lesser inhibitory effects against the same enzymes insolubilized. Further, presentation of Gtf antigen immobilized to HA has only a minor influence on the production of antibodies inhibitory to Gtf activity.