Identification of a fliG homologue in Treponema denticola.

Using a bacteriophage lambda library of Treponema denticola (Td) ATCC 35405 DNA, and, as a reagent, sera derived from individuals with advanced adult periodontal disease, a variety of recombinant clones producing antigens of this oral spirochete have been isolated. Nucleotide sequence analysis of a clone expressing three immunoreactive antigens has revealed the presence of an open reading frame highly homologous to the flagellar switch/motor protein, FliG, which is known to be essential for flagellar assembly and rotation, and chemotaxis in enteric bacteria. The deduced amino-acid sequence of the treponemal FliG protein had 73% similarity (55% identity) to the Bacillus subtilis FliG protein, and showed significant, but lesser homologies to Gram- FliG proteins. Sequence analysis of regions flanking fliG indicated that this gene is immediately preceded by a fliF homologue, further supporting that the cloned DNA encodes FliG of Td. The findings imply that although the signals for control of chemotaxis may be distinctly different in spirochetes, at least some of the molecules involved in torque generation, control of flagellar rotation and signal transduction are highly conserved with other bacteria. The stronger homology of the spirochete FliG with those of Gram+ bacteria is also consistent with recent analyses of other spirochetal genes.

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA.

Streptococcus mutans GS-5 synthesizes an exo-beta-D-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans. These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase.

The complete nucleotide sequence (5,010 bp) of the fructanase gene (fruA) and flanking regions of the chromosome of Streptococcus mutans GS-5 was determined. The fruA gene appears to be the sole transcript arising from a proximal promoter. The presumed precursor of the secreted FruA protein consists of 1,423 amino acids, and it has an M(r) of 158,656 and a pI of 4.82. The N terminus of FruA has characteristics in common with signal peptides of gram-positive organisms. The C terminus consists of a serine- and threonine-rich region, followed by the peptide LPDTGD, 4 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the wall-spanning region, the LPXTGX consensus sequence, and the membrane-spanning domains of surface-associated proteins of gram-positive cocci. The FruA protein has significant homology with the Bacillus subtilis levanase (SacC), the Bacteroides fragilis levanase (ScrL), yeast invertases, and a number of other beta-fructosidases but not with fructosyltransferase, glucosyltransferases, or glucan-binding proteins of oral streptococci. Genes with homology to fruA were detected in S. mutans serotype c, e, and f strains, Streptococcus rattus, Streptococcus salivarius, and Streptococcus sanguis. A deletion derivative of FruA lacking the C-terminal 437 amino acids was still functional and could hydrolyze beta-(2,6)- and beta-(2,1)-linked sugars, but with altered preference for substrates. The data begin to define functional domains of the FruA protein and potential regulatory sites for induction, repression, growth rate control, and posttranslational localization of this multifunctional enzyme.

Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification.

The dental pathogen Streptococcus mutans persists and causes diseases in highly dynamic environments and gains a selective ecological advantage in environmental conditions that frequently exceed the limits for growth of the organism, particularly with regard to environmental pH. The goal of this study was to begin a molecular genetic analysis of a major stress protein, DnaK/Hsp70, to begin to understand how stress responses are regulated in this lactic acid bacterium and to establish a relationship between dnaK gene expression and exposure to acidic environments. Cloning and nucleotide sequence analysis revealed that the dnaK gene is preceded by, and is in an operon-like arrangement with, the hrcA and grpE genes, although intergenic spacing was unlike that described in other bacteria. An inverted repeat (a CIRCE element) was identified by sequence analysis and, using primer extensions, a heat shock-responsive, sigmaA-type promoter, P1, 5' to the hrcA gene, and a sigmaB-type promoter, 5' to the grpE translational start site, were identified. No promoters were detected between grpE and dnaK. A strain carrying a strongly polar insertion in the hrcA gene had markedly diminished levels of dnaK mRNA, indicating that dnaK was transcribed as part of an operon from P1, and to a lesser extent from P2. Results from physiological manipulation of S. mutans in continuous chemostat culture demonstrated that steadystate levels of S. mutans dnaK mRNA and DnaK protein were (i) increased in response to acid shock; (ii) elevated in acid 'adapted' cells; and (iii) induced in response to alkali shock of acid 'adapted' cells. In all cases, increased amounts of dnaK mRNA could be correlated with enhanced transcription from P1. This study provides the first detailed analysis of the expression of a heat shock gene from an oral isolate, and the evidence provided suggests that sigmaB-like promoters may also be involved in class I heat shock gene expression in some Gram-positive organisms.

Analysis of gene expression in Streptococcus mutans in biofilms in vitro.

The purpose of this study was to develop methods for the consistent production of biofilms of S. mutans containing reporter gene fusions, and to examine the expression of genes involved in sucrose metabolism in adherent populations of this organism. Three strains of S. mutans harboring reporter gene fusions to the gene promoter regions of the gtfBC genes, ftf, and scrA were grown in a Rototorque biofilm fermenter in a tryptone-yeast extract-sucrose medium. Quasi-steady-state levels of reporter gene activity were measured after the biofilms were grown for either 48 hrs of 7 days. Also, induction of gene expression by the addition of sucrose to biofilm cells was monitored. Reporter gene activity was measurable from all gene fusion strains. This study (i) establishes the feasibility of doing detailed molecular and physiologic studies on immobilized populations of S. mutans, (ii) demonstrates that the polysaccharide synthesis machinery of S. mutans is differentially expressed in biofilms, and (iii) opens the way for a more detailed analysis of the environmental signals and signal transduction pathways governing the regulation of gene expression by S. mutans cells that are immobilized on a solid surface.

Characteristics and cariogenicity of a fructanase-defective Streptococcus mutants strain.

Polymers of D-fructose produced by a variety of oral bacteria are believed to function as extracellular carbohydrate reserves. Degradation of these polysaccharides in plaque following exhaustion of dietary carbohydrates is thought to contribute to the extent and duration of the acid challenge to the tooth surface and thus to the initiation and progression of dental caries. Streptococcus mutans produces a fructanase, the product of the fruA gene, which is capable of degrading beta(2,6)- and beta(2,1)-linked fructans that are commonly synthesized by dental plaque microorganisms. To evaluate the role of the FruA protein in exopolysaccharide metabolism and to assess the contribution of this enzyme to the pathogenic potential of S. mutans, a fructanase-deficient strain of S. mutans was constructed. Inactivation of a cloned fruA gene was accomplished in Escherichia coli by using a mini-Mu dE transposon, and then an isogenic mutant of S. mutans UA159 was constructed by allelic exchange. Successful inactivation of fruA was confirmed through the use of biochemical assays, Western blotting (immunoblotting) with anti-recombinant FruA antisera, and Southern hybridization. The data indicated that FruA was the only fructan hydrolase produced by S. mutans UA159. Inactivation of fruA had no significant effects on glucosyltransferase or fructosyltransferase activity. In the rat caries model using animals fed a high-sucrose diet and ad libitum, there were no significant differences in the number or severity of smooth surface, sulcal, or root caries elicited by the fruA mutant and the wild-type organism.

Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression.

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction.