OBJECTIVEThe purpose of this study is to construct a pyruvate oxidase gene deficiency variant strain of Streptococcus oralis (S. oralis).

METHODSThe sopox gene, which was got using polymerase chain reaction (PCR), and the 130-basepair segment of which was cut down with endonuclease BamHI, and transferred into S. oralis (ATCC10557) by using electrotransformation. The authors obtained a variant strain of S. oralis, and then the catalase activity of the first culture and 3-4 subcultures was examined.

RESULTSThe authors obtained a pyruvate oxidase gene deficiency variant strain of S. orlis. The catalase activity examination showed that the ability of producing H2 O2 of the variant strain of S. orlis declined, whose catalase activity was between those of the positive control (ATCC10557) and the negative control (Escherichia coli, JM109). But the produced H2 O2 quantity of their subcultures was less than that of the negative control.

CONCLUSIONThe construction of the pyruvate oxidase gene deficiency variant strain of Streptococcus oralis is successful.

Cloning, Molecular ; Genes, Bacterial ; Genetic Engineering ; Genetic Variation ; Polymerase Chain Reaction ; Pyruvate Oxidase ; genetics ; Streptococcus oralis ; enzymology ; genetics

OBJECTIVETo elucidate the molecular structure of pyruvate oxidase gene promoter.

METHODSThe 1.30 kb fragment with promoter activity, amplified from upstream of Streptococcus oralis pyruvate oxidase gene (Sopox), was cloned into vector PBK-CMV. The positive transformed E. coli JM109 clone was selected, the recombinant plasmid was further identified with restriction mapping analysis. The positive recombinant plasmid was studied with sequence analysis.

RESULTSAfter digesting the recombinant plasmid with Hind III, 1% agarose electrophoresis showed 1.30 kb fragment, which was consistent with predicted size. Sequence analysis revealed 1,350 bp.

CONCLUSIONThe Sopox promoter region is sequenced. Further characterization of the Sopox promoter region will elucidate the molecular mechanism of H2O2 production of streptococcus oralis.

Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; Genes, Bacterial ; Humans ; Hydrogen Peroxide ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Pyruvate Oxidase ; genetics ; Sequence Analysis, DNA ; Streptococcus oralis ; enzymology ; genetics

The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase (SpxB) gene expression and protein production in Streptococcus sanguinis (S. sanguinis). SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide (H2O2) able to antagonize cariogenic Streptococcus mutans (S. mutans). Furthermore, the ecological consequence of H2O2 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H2O2, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H2O2 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production.
Antibiosis ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Epitopes ; genetics ; Gene Expression Regulation, Bacterial ; Hydrogen Peroxide ; metabolism ; pharmacology ; Oligopeptides ; Oxygen ; metabolism ; Peptides ; genetics ; Pyruvate Oxidase ; biosynthesis ; genetics ; Streptococcus mutans ; drug effects ; Streptococcus sanguis ; enzymology ; genetics ; growth & development ; Transformation, Bacterial