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Novel Sucrose-dependent Adhesion Co-factors in Streptococcus mutans

¹ Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, IL 60612; and
² Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030;

View larger version (83K): [in a new window]   Figure 1. Phenotypic analysis of S. mutans sucrose-dependent adhesion mutants. (A) Comparison of S. mutans wild-type and mutant adhesion to microtiter plate wells. Well number: 1, LT11 (wt); 2, LT41; 3, LT42; 4, LT43; and 4, LT44. Bacteria were grown at 37°C in 0.2 mL of Jordan medium supplemented with 3% sucrose in a sterile polystyrene microtiter plate. After 48 hrs, culture liquor was removed, and wells were washed three times with de-ionized water. The adherent bacteria were stained with 1% crystal violet for 1 min, rinsed with de-ionized water, and photographed. (B) Coomassie Blue stain of SDS-PAGE of S. mutans extracellular proteins. Lanes: M, molecular-weight standards; 1 (LT11); 2 (LT41); 3 (LT42); 4 (LT43); and 5 (LT44). (a) Surface proteins in culture liquor (20 X concentrated); (b) surface proteins released by sonication; and (c) surface proteins not released by sonication. Little surface protein was released from LT41 by sonication (lane b2). The sample buffer elution (lane c5) released a band of 160 kD (arrows) from LT44 that was weakly evident in the culture liquor (lane a5), and not evident after sonication (lane b5). (C) Periodic acid-Schiff's stain of SDS-PAGE of the same S. mutans samples. Two polysaccharide bands at the 150-kD and 90-kD levels, corresponding to Gtf and fructosyltransferase (Ftf) activities, respectively, are similar between LT11 and four mutants in the cultural liquor fraction. There was no noticeable Gtf or Ftf activity in the sonication-released fraction for LT41. Although little protein was released (Panel B) from LT43 by either sonication (b4) or sample buffer (c4), its free Gtf and Ftf activities (Panel C, a4) appeared strong.

View larger version (16K): [in a new window]   Figure 2. Structural organization of the four loci encoding sucrose-dependent adhesion co-factors of S. mutans. The triangle indicates the insertion site of pVA891. (A) The locus in LT41, a putative glycerol-3-phosphate dehydrogenase (gpdA). Downstream of gpdA, separated by one base, is gluA (UDP-glucose-1-phosphate uridylyltransferase; Yamashita et al., 1998). Upstream gpdA locate two divergent putative promoters and two ORFs on the opposite strand. The first ORF encodes a putative dUTPase (dutA). (B) The locus in LT42, a putative ABC transporter located between two other ABCs. On the left locates an operon of five genes for amino acid transport and metabolism (atm). On the right is a protein-secreting ABC (psa) transporter of three genes (Ross et al., 1993). The function of the central (mutated) ABC is unknown. Six genes had homologies to known genes: cnhA (carbon-nitrogen hydrolase), gidB (glucose-inhibited division protein), htpX (heat-shock protease), lemA (lemA-like protein), ylbN (YibN-like protein), and gcrR (regulator of Gbp-C; Sato et al., 2000). (C) The locus in LT43, a putative multidrug efflux pump (mepA). A transcriptional regulator was found upstream, while downstream mepA is gbpA separated by a small ORF on a separate strand. (D) The locus in LT44, the putative ribulose monophosphate operon. It encodes three PTS enzyme II components (rmpABC), a probable hexulose-6-P synthase (rmpD), a hexulose-6-P isomerase (rmpE), and a ribulose-5-P-4 epimerase (rmpF).