Identification of A2059G 23S rRNA and G439A rplC gene mutations in Streptococcus criceti strain OMZ 61, a strain resistant to azithromycin, josamycin and clindamycin.

Streptococcus criceti is a cariogenic organism that belongs to the mutans streptococci. Of the four S. criceti strains, strain OMZ 61 has been identified as being resistant to erythromycin. Antimicrobial susceptibility testing showed that strain OMZ 61 is also resistant to azithromycin, josamycin and clindamycin but susceptible to tetracycline and tiamulin. DNA hybridization analysis of the 23S rRNA genes revealed that the hybridization patterns in strain OMZ 61 differed from those in the other three strains. We further analyzed the nucleotide sequences of a ribosomal RNA operon, the rrnD operon, and the rpsJ-rpsQ region including rplC and rplD genes for ribosomal proteins L3 and L4, respectively, in the four strains studied. Nucleotide sequence analysis indicated that strain OMZ 61 contains an A-to-G substitution at nucleotide position 2059, equivalent to Escherichia coli numbering 2058, in a 23S rRNA gene (rrlD) and a G-to-A substitution at nucleotide position 439 in the rplC gene, suggesting an amino acid residue change at position 147 from valine to isoleucine, whereas no mutation in the rplD gene was found. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism analysis showed that most or all of the 23S rRNA genes in strain OMZ 61 contain the A2059G mutation. These findings suggest that the resistance to erythromycin, azithromycin, josamycin and clindamycin in strain OMZ 61 is conferred by alterations in 23S rRNA and/or ribosomal protein L3. This is the first description of mutations in the 23S rRNA and rplC genes in mutans streptococci.

Molecular characterization of the dextran-binding lectin B gene dblB of Streptococcus criceti in Streptococcus mutans strain GS-5 with mutations in both gbpC and spaP genes.

Streptococcus mutans, a cariogenic agent, has a glucan-binding protein gene, gbpC, and S. criceti possesses four gbpC homologs, including dblA and dblB, as does S. sobrinus. The S. criceti dblB gene encodes a 1,717-amino-acid protein having two repetitive alanine-rich and proline-rich regions and an LPXTG motif, which is recognized by the sortase SrtA, near the C terminus. Reverse transcription-PCR analysis indicated no cotranscription of the dblA and dblB genes of S. criceti. As we could not obtain a dblB mutant of S. criceti, the dblB gene was characterized in S. mutans strain GS-5, which has genetic mutations in both gbpC and spaP genes and shows an inability to agglutinate triggered by dextran. A dextran-induced agglutination assay showed that S. mutans cells carrying dblB agglutinated in the presence of dextran. A hydrophobicity assay showed that the cells containing dblB were hydrophobic. A biofilm formation assay showed that the dblB gene was associated with biofilm formation by cells cultivated in brain heart infusion broth supplemented with glucose and maltose, but not sucrose. Nucleotide sequence analysis of the S. criceti strains studied revealed a frameshift mutation in the srtA gene encoding sortase, but intact dblA and dblB genes were found in dextran-induced agglutination-negative strains, whereas intact dblA, dblB and srtA genes were found in dextran-induced agglutination-positive strains. These results suggest the cell-surface localization of dblA and dblB gene products by SrtA and the responsibility of dblB for dextran-induced agglutination, cell-surface hydrophobicity and biofilm formation in S. criceti.

Identification and characterization of an autolysin gene, atlA, from Streptococcus criceti.

AtlA of Streptococcus mutans is a major autolysin and belongs to glycoside hydrolase family 25 with cellosyl of Streptomyces coelicolor. The autolysin gene (atlA) encoding AtlA was identified from S. criceti. AtlA of S. criceti comprises the signal sequence in the N-terminus, the putative cell-wall-binding domain in the middle, and the catalytic domain in the C-terminus. Homology modeling analysis of the catalytic domain of AtlA showed the resemblance of the spatial arrangement of five amino acids around the predicted catalytic cavity to that of cellosyl. Recombinant AtlA and its four point mutants, D655A, D747A, W831A, and D849A, were evaluated on zymogram of S. criceti cells. Lytic activity was destroyed in the mutants D655A and D747A and diminished in the mutants W831A and D849A. These results suggest that Asp655 and Asp747 residues are critical for lytic activity and Trp831 and Asp849 residues are also associated with enzymatic activity.

Characterization of Streptococcus criceti insertion sequence ISScr1.

A novel insertion sequence element of the IS982 family, ISScr1, was previously identified in Streptococcus criceti strain E49 as a disrupted paaB gene encoding an antigen I/II homologous protein. In this study, we identified two divergent inserted regions of ISScr1 in S. criceti E49 by inverse polymerase chain reaction, the gene-walking method, and screening of the partial plasmid library. Nucleotide sequence analysis indicated the possible explanation that transposition generated 8- and 9-bp direct repeat sequences. DNA hybridization analysis revealed that an identical hybridization pattern to ISScr1 was observed in the four S. criceti strains studied and that at least three copies of ISScr1 were preserved in S. criceti strains. In addition, we found different susceptibility to erythromycin and diverse agglutination properties induced by dextran in S. criceti. Furthermore, DNA hybridization analysis showed that no ISScr1-like copy was detected in the other 14 strains of oral streptococci tested.

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H(2)S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H(2)S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H(2)S from L-cysteine (approximately 30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing betaC-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced H(2)S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H(2)S in distinct manners: Fn0625 carries out beta-elimination of L-cysteine to produce H(2)S, pyruvate and ammonia, whereas Fn1220 catalyses the beta-replacement of L-cysteine to produce H(2)S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

Use of a novel assay to evaluate enzymes that produce hydrogen sulfide in Fusobacterium nucleatum.

To evaluate enzymes that produce hydrogen sulfide (H(2)S) in species of Fusobacterium nucleatum, we developed an assay based on SDS-polyacrylamide gel electrophoresis with renaturation followed by active staining. This assay provided precise insight into the enzymes that produce H(2)S in terms of their number and molecular weights.

Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis.

Indole produced via the beta-elimination reaction of l-tryptophan by pyridoxal 5'-phosphate-dependent tryptophanase (EC 4.1.99.1) has recently been shown to be an extracellular and intercellular signalling molecule in bacteria, and controls bacterial biofilm formation and virulence factors. In the present study, we determined the molecular basis of indole production in the periodontopathogenic bacterium Porphyromonas gingivalis. A database search showed that the amino acid sequence deduced from pg1401 of P. gingivalis W83 is 45 % identical with that from tnaA of Escherichia coli K-12, which encodes tryptophanase. Replacement of the pg1401 gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction of pg1401, demonstrating that the gene is functionally equivalent to tnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlike E. coli tnaA, pg1401 is expressed alone in P. gingivalis and that the nucleotide sequence of the transcription start site is different, suggesting that the expression of P. gingivalis tnaA is controlled by a unique mechanism. Purified recombinant P. gingivalis tryptophanase exhibited the Michaelis-Menten kinetics values K(m)=0.20+/-0.01 mM and k(cat)=1.37+/-0.06 s(-1) in potassium phosphate buffer, but in sodium phosphate buffer, the enzyme showed lower activity. However, the cation in the buffer, K(+) or Na(+), did not appear to affect the quaternary structure of the enzyme or the binding of pyridoxal 5'-phosphate to the enzyme. The enzyme also degraded S-ethyl-l-cysteine and S-methyl-l-cysteine, but not l-alanine, l-serine or l-cysteine.

Identification and characterization of an autolysin gene, atlh, from Streptococcus downei.

An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.

Identification and characterization of an autolysin gene, atlg, from Streptococcus sobrinus.

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans. In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA-inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA-deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus.

Identification and molecular analysis of betaC-S lyase producing hydrogen sulfide in Streptococcus intermedius.

Hydrogen sulfide (H(2)S) is a toxic gas that induces the modification and release of haemoglobin in erythrocytes; however, it also functions in methionine biosynthesis in bacteria. betaC-S lyase, encoded by the lcd gene, is responsible for bacterial H(2)S production through the cleavage of l-cysteine. In this study, 26 of 29 crude extracts from reference and clinical strains of Streptococcus intermedius produced H(2)S from l-cysteine. The capacities in those strains were not higher than those in strains of the other anginosus group of streptococci, Streptococcus anginosus and Streptococcus constellatus, but were much greater than those in strains of Streptococcus gordonii, which is known to have an extremely low capacity for H(2)S production. Incubation of the remaining three extracts with l-cysteine did not result in H(2)S production. Sequence analysis revealed that the lcd genes from these three strains (S. intermedius strains ATCC 27335, IMU151 and IMU202) contained mutations or small deletions. H(2)S production in crude extracts prepared from S. intermedius ATCC 27335 was restored by repairing the lcd gene sequence in genomic DNA. The kinetic properties of the purified recombinant protein encoded by the repaired lcd gene were comparable to those of native proteins produced by H(2)S-producing strains, whereas the truncated protein produced by S. intermedius ATCC 27335 had no enzymic activity with l-cysteine or l-cystathionine. However, real-time PCR analysis indicated that the lcd gene in strains ATCC 27335, IMU151 and IMU202 is transcribed and regulated in a manner similar to that in the H(2)S-producing strain.

Identification and characterization of an antigen I/II homologous gene, pah, from Streptococcus downei.

Antigen I/II of Streptococcus mutans is a cell surface protein involved in the adherence of cells to tooth surfaces. In this study, an antigen I/II homologous gene, pah, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. The pah gene encodes a cell-wall-anchoring protein, PAh, containing 1565 amino acids. At the deduced amino acid sequence level, PAh shows a strong similarity to PAg of S. sobrinus (97.6% identity). Southern hybridization analysis indicated that a single copy of the pah gene was preserved in the chromosomal DNA of S. downei. Two pah mutants, SES-1 and SES-2, were constructed and analyzed by Western blotting. Two bands corresponding to 200- and 160-kDa proteins were observed in the parent strain, whereas no band was detected in pah mutant strains. In an adhesion assay of cells, pah mutants failed to adhere to tube surfaces in contrast to the parent strain. Furthermore, saliva-induced aggregation was decreased in pah mutants compared to the parent strain. Together, PAh is associated with the adhesion of cells to abiotic surfaces and whole saliva.

Identification and characterization of a dextranase gene of Streptococcus criceti.

The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity.

An improved method for assaying phosphatidylcholine in mouse tissue.

INTRODUCTION: To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an alpha1-adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo. METHODS: Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 degrees C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay. RESULTS: The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% (n = 10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% (n = 10) and 1.3% (n = 30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 micromol/g (eye ball) and 29 micromol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland. DISCUSSION: Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.

Identification of another surface protein antigen I/II gene, paaB, and a putative transcriptional regulator gene, par, from Streptococcus cricetus.

The flanking region of the antigen I/II gene, paaA, in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, ISScr1. ISScr1 is a member of the IS982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene (pag) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus. Therefore, ORF3 was designated the par gene of S. cricetus. Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.