OBJECTIVETo detect and analyze two important genes, comE and luxS, in quorum sensing signal pathway from Streptococcus oralis (S. oralis).

METHODSThe total genomic DNA of S. oralis NH521 (a clinically isolated strain) was firstly extracted. The comE and luxS genes were then amplified by polymerase chain reaction (PCR) and further sequenced. The obtained sequences were compared with related sequences in GenBank.

RESULTSTarget bands of both comE and luxS genes were detected by electrophoresis. The obtained gene sequences were similar to the corresponding sequences from another S. oralis strain (luxS, 95.0%; comE, 99.6%); however, comparing to gene sequences of another species Streptococcus mutans, comE was more divergent (12.7%) than luxS gene (74.1%).

CONCLUSIONThis study successfully amplified and sequenced comE and luxS genes from S. oralis NH521 strain. The luxS gene accumulated more mutations than comE gene did between two S. oralis strains, but comE gene is more divergent than luxS gene between two Streptococcus species.

Bacterial Proteins ; Carbon-Sulfur Lyases ; Polymerase Chain Reaction ; Quorum Sensing ; Signal Transduction ; Streptococcus mutans ; Streptococcus oralis

Bacterial Proteins ; metabolism ; Carbon-Sulfur Lyases ; metabolism ; Mouth ; microbiology ; Quorum Sensing ; Signal Transduction

OBJECTIVETo explore the mechanism of virulence regulation in group B streptococcus (BGS) by studying LuxS-related AI-2 quorum-sensing pathway in GBS.

METHODSluxS gene deletion mutants of GBS (Delta lusX) were characterized by reverse transcription (RT)-PCR, colony immunoblot analysis, growth curve measurement, and cAMP determination. Functional analysis of luxS in the deletion mutants was conducted by bioluminescence assay.

RESULTSGenetic analysis results showed that the luxS deletion in the mutant 515-Delta lusX caused upregulation of scpB gene expression. Phenotypic analysis revealed that, in comparison with the wild strain, 515-Delta lusX mutant grew slowly in DCM media but quickly in THY media. An approximately two-fold decrement in bioluminescence was detected in the luxS deletion mutants as compared with the wild strain.

CONCLUSIONThis study confirms the importance of LuxS molecule in the AI-2 quorum-sensing pathway in GBS and provides new insights into the virulence regulation mechanism of GBS.

Bacterial Proteins ; genetics ; Carbon-Sulfur Lyases ; genetics ; Gene Deletion ; Gene Expression Regulation, Bacterial ; Lighting ; Phenotype ; Streptococcus agalactiae ; genetics ; pathogenicity ; Virulence

OBJECTIVETo construct luxS mutant aften luxS gene of Streptococcus mutans (S. mutans) was knocked out, and examine their ability of biofilm formation.

METHODSA recombinant plasmid containing the flanking fragment of luxS of S. mutans was transformed into S. mutans UA159, and selected by brain heart infusion (BHI) agar medium with kanamicin. The luxS mutant further confirmed via polymerase chain reaction (PCR) and the autoinducer-2 (AI-2) bioluminescence assay of Vibrio harveyi (V. harveyi), and ability of luxS mutant and S. mutans UA159 biofilm formation was examined in different phases, in BHI medium with 1% sucrose and 1% glycose by scanning electron microscopy (SEM).

RESULTSLuxS-deficient S. mutans strains were successfully constructed. Compared with S. mutans UA159, the luxS mutant maintained in BHI medium containing 1% sucrose displayed an apparent defect in biofilm formation, while they showed no significant deviation in BHI medium containing 1% glycose.

CONCLUSIONluxS gene in S.mutans can play a role in dental plaque biofilm formation, and the luxS gene is possible to regulate sucrose-dependent biofilm formation.

Bacterial Proteins ; Biofilms ; Carbon-Sulfur Lyases ; Culture Media ; Dental Plaque ; Homoserine ; analogs & derivatives ; Humans ; Lactones ; Streptococcus mutans

OBJECTIVETo investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans.

METHODSThe differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm.

RESULTSThe transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05).

CONCLUSIONSThe methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.

Bacterial Proteins ; metabolism ; Biofilms ; Carbon-Sulfur Lyases ; metabolism ; Glucosyltransferases ; Microscopy, Confocal ; Real-Time Polymerase Chain Reaction ; Streptococcus mutans ; metabolism

OBJECTIVEIt is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid.

METHODSThe upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially.

RESULTSWith endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro.

CONCLUSIONThe S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.

Bacterial Proteins ; Carbon-Sulfur Lyases ; Escherichia coli ; Genetic Vectors ; Plasmids ; Polymerase Chain Reaction ; Quorum Sensing ; Streptococcus mutans

Methionine-dependent increase in tumor cells is a specific metabolic defect. This metabolic defect is also a target for selective treatment of cancer. Studies found that the methionine gamma-lyase (methioninase, L-methionine gamma-lyase) can specificly split the methionine of extracellular and intracellular, so it can strongly inhibit the growth of tumor cells and induce apoptosis of tumor cells. However, no effect on normal cells has been found, so the methionine gamma-lyase may play the anti-tumor role. We also explored in the present study another effect of methionine gamma-lyase as a single agent on DNA methylation levels and DNA synthesis, which may change as a result of deprivation of methionine, and thus may enhance anti-tumor effects. Animal studies and clinical trials showed that a variety of methionine dependent methionine gamma-lyase can eliminate the tumor cells. Therefore, methionine restriction is an effective anticancer strategy. Methionine gamma-lyase has shown great prospects as a new type of gene therapy. This article made a review of it.
Animals ; Carbon-Sulfur Lyases ; administration & dosage ; Genetic Therapy ; methods ; Humans ; Neoplasms ; drug therapy ; therapy ; Recombinant Proteins ; administration & dosage ; Transfection

OBJECTIVETo detect the AI-2 quorum-sensing pathway and construct the luxS g-ene allelic exchange plasmid of Streptococcus mutans.

METHODSTo detect AI-2 pathway in Streptococcus mutans, the Vibrio harveyi BB170 was used as reporter strain. The PCR fragments of the upstream and downstream regions of luxS and the Erythromycin resistance gene were amplified with the primers respectively, and these fragments were ligated into pUC19 vector with double endonuclease reaction sequentially, the ligated DNAs were transformed into Escherichia coli DH5alpha, then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions.

RESULTSStreptococcus mutans Ingbritt C could induce luminescence of BB170, suggesting the presence of AI-2 quorum sensing pathway in Streptococcus mutans, and such stimulatory activity was maximal at the mid-log growth phase. The recombinant plasmid pUCluxKO was digested by PstI-BamHI, and the digest product were 1000 bp and 5000 bp. When the pUCluxKO was digested by BamHI-KpnI, the digest product were 1500 bp and 4500 bp. While it was digested by KpnI-EcoRI, the digest product were 1000 bp and 5000 bp. All PCR product was in a single belt respectively.

CONCLUSIONSThe recombinant plasmid was cloned effectively and can be used in the construction of S.mutans luxS mutant.

Bacterial Proteins ; genetics ; Carbon-Sulfur Lyases ; genetics ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; Homoserine ; genetics ; Plasmids ; Quorum Sensing ; genetics ; Streptococcus mutans ; genetics

OBJECTIVETo investigate the effect of Streptococcus mutans luxS mutarotation on the early biofilm formation.

METHODSBased on the immobilization of magnetic beads by adherent cells, an assay of biofilm quantitative analysis was developed for the kinetic quantification of biofilm formation in this study. Streptococcus mutans luxS mutant strain was constructed and subject to this biofilm luxS mutant strain were compared.

RESULTSThe delta luxS mutant started to form a biofilm from the 6th hour (delta BFI = 2.015), and the delta BFI of luxS mutant increased more quickly than that of the wild type strain, until reaching a complete immobilization of the beads after 10 hours (delta BFI = 7.025). The wild-type strain start to form a biofilm from the 10 th hour (delta BFI = 1.875) and the beads were completely immobilized between 12 and 14 hours.

CONCLUSIONSThe luxS mutation can accelerate biofilm on a polystyrene surface during the mid-exponential growth phase. And a luxS-dependent signal may play an important role in the early biofilm formation of Streptococcus mutans.

Bacterial Proteins ; genetics ; Biofilms ; growth & development ; Carbon-Sulfur Lyases ; genetics ; Gene Deletion ; Gene Expression Regulation, Bacterial ; Streptococcus mutans ; genetics ; growth & development

OBJECTIVETo detect the presence and distribution of luxS gene in quorum sensing signal system in the periodontal pathogens.

METHODSThe total DNA of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), Actinobacillus acitinomycetimcomtans (Aa) were extracted. The presence of luxS was detected by polymerase chain reaction (PCR). The products of PCR were detected by electrophoresis, sequenced and identified by a Blast search of the GenBank database.

RESULTSElectrophoresis, sequencing and Blast searching indicated that the PCR products of Pg were highly consistent with the luxS gene in GenBank. The sequencing result of Fn was also identified with the target gene. The PCR product of Aa was the same as reference through electrophoresis.

CONCLUSIONSPg, Fn, Aa contain luxS gene. Further studies may be required to investigate the functions of luxS in the periodontal pathogens.

Aggregatibacter actinomycetemcomitans ; genetics ; metabolism ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Carbon-Sulfur Lyases ; genetics ; isolation & purification ; metabolism ; Fusobacterium nucleatum ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Porphyromonas gingivalis ; genetics ; metabolism ; Quorum Sensing ; genetics ; Signal Transduction