A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1^– mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Le^a(Gal1,3(Fuc1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Le^b, Le^x, Le^y, H1, H2, A, B and sialylated Le^a did not. Le^a without galactose (Fuc1,4GlcNAc) still bound to S. mutans, but Le^a without fucose (Gal1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le^a. In conclusion, S. mutans can bind to Le^a carbohydrate epitopes in which the fucose is an essential residue. Le^a carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.     

Streptococcal-host interactions. Structural and functional analysis of a Streptococcus sanguis receptor for a human salivary glycoprotein

Colonization of oral tissues by Streptococcus sanguis may be influenced by a mucin-like salivary glycoprotein (SAG) through a calcium-dependent interaction with a specific bacterial receptor. We report the nucleotide and deduced amino acid sequence of the S. sanguis receptor (SSP-5) and show that this protein may bind sialic acid residues of SAG. The SSP-5 protein contains three unique structural domains, two of which consist of repetitive amino acid sequences. The N-terminal domain is comprised of four tandem copies of an 82-residue repeat which exhibits homology to M protein of Streptococcus pyogenes. This region is highly charged and predicted to be alpha-helical. A second hydrophilic repetitive domain consists of three copies of a 39-amino acid sequence containing 30% proline flanked by nonrepetitive proline-rich sequence. The third domain consists of 48% proline and resides near the C terminus of the protein. Secondary structure analysis of the SSP-5 sequence also identified four potential helix-turn-helix motifs that resembled E-F hand calcium binding domains. The SSP-5 protein is highly homologous to a surface antigen expressed by the mutans streptococci and the domain structure of SSP-5 is conserved within this family of proteins. The interactions of SSP-5 and of intact S. sanguis with SAG were inhibited by neuraminidase digestion of the salivary glycoprotein and by simple sugars containing sialic acid, suggesting that sialic acid is the primary ligand involved in the binding reaction.     

SMU.746-SMU.747, a Putative Membrane Permease Complex,Is Involved in Aciduricity,Acidogenesis, and Biofilm Formation in Streptococcus mutans

Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium''s ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F₁F₀ ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.     

Genetic identification of a locus linked to the rat MHC that codes for a membrane antigen detectable with cytotoxic lymphocytes.

Cell-mediated cytolytic (CMC) responses resulted from immunizations between rat strains purported to be identical at Ag-B, the major histocompatibility complex (MHC), but differing at other loci not linked to Ag-B. In vivo-priming followed by secondary in vitro stimulation was required to generate a measurable CMC response as determined by a 51Cr-release assay. Neither in vivo nor in vitro stimulation alone was adequate. The CMC responses generated in a strain combination considered Ag-B identical (LEW . B3:BN) were specific for a determinant controlled by a gene linked to Ag-B, which has been designated Ag-L. The CMC response appears not to be restricted to sygeneity at Ag-B. In addition, the data presented demonstrate a recombinant between Ag-B and Ag-L, and suggest that the gene has failed to transfer with the MHC during the isolation of the LEW . B3 and F-344 . B3 congenic strains.     

Identification of genes that interact with glp-1, a gene required for inductive cell interactions in Caenorhabditis elegans

The glp-1 gene functions in two inductive cellular interactions and in development of the embryonic hypodermis of C. elegans. We have isolated six mutations as recessive suppressors of temperature-sensitive (ts) mutations of glp-1. By mapping and complementation tests, we found that these suppressors are mutations of known dumpy (dpy) genes; dpy genes are required for development of normal body shape. Based on this result, we asked whether mutations previously isolated in screens for mutants defective in body shape could also suppress glp-1(ts). From these tests, we learned that unselected mutations of eight genes required for normal C. elegans morphogenesis, including the four already identified, suppress glp-1(ts). All of these suppressors rescue all three mutant phenotypes of glp-1(ts) (defects in embryonic induction of pharyngeal tissue, in embryonic hypodermis development, and in induction of germline proliferation). However, they do not rescue putative glp-1 null mutants and therefore do not bypass the requirement for glp-1 in development. In the light of current ideas about the molecular nature of the glp-1 and suppressor gene products, we propose an interaction between the glp-1 protein and components of the extracellular matrix and speculate that this interaction may impose spatial constraints on the decision between mitosis and meiosis in the germline.     

Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.     

Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system

The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.     

Biochemical and molecular characterization of a novel type of Mutanase from Paenibacillus sp. strain RM1: identification of its mutan-binding domain, essential for degradation of Streptococcus mutans biofilms

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.     

Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism.

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.     

Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha -UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC

The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-beta(1,4)-GlcNAc-alpha(1- and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis. Previously, the KfiC protein was shown to be a beta-UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an alpha-UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA. In addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.     

Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase.

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca(2+)-mediated signaling in many cells. Yeast cells lacking functional calcineurin (cna1 cna2 or cnb1 mutants) display growth defects under specific environmental conditions, for example, in the presence of high concentrations of Na+, Li+, Mn2+, or OH- but are indistinguishable from wild-type cells under standard culture conditions. To characterize regulatory pathways that may overlap with calcineurin, we performed a synthetic lethal screen to identify mutants that require calcineurin on standard growth media. The characterization of one such mutant, cnd1-8, is presented. The CND1 gene was cloned, and sequence analysis predicts that it encodes a novel protein 1,876 amino acids in length with multiple membrane-spanning domains. CND1 is identical to the gene identified previously as FKS1, ETG1, and CWH53, cnd1 mutants are sensitive to FK506 and cyclosporin A and exhibit slow growth that is improved by the addition of osmotic stabilizing agents. This osmotic agent-remedial growth defect and microscopic evidence of spontaneous cell lysis in cnd1 cultures suggest that cell integrity is compromised in these mutants. Mutations in the genes for yeast protein kinase C (pkc1) and a MAP kinase (mpk1/slt2) disrupt a Ca(2+)-dependent signaling pathway required to maintain a normal cell wall and cell integrity. We show that pkc1 and mpk1/slt2 growth defects are more severe in the absence of calcineurin function and less severe in the presence of a constitutively active form of calcineurin. These observations suggest that calcineurin and protein kinase C perform independent but physiologically related functions in yeast cells. We show that several mutants that lack a functional vacuolar H(+)-ATPase (vma) require calcineurin for vegetative growth. We discuss possible roles for calcineurin in regulating intracellular ion homeostasis and in maintaining cell integrity.     

Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth

Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity.     

Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.

In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.