RESUMO
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.
Assuntos
Antibiose/fisiologia , Proteínas de Bactérias/biossíntese , Piruvato Oxidase/biossíntese , Streptococcus mutans/efeitos dos fármacos , Streptococcus sanguis/enzimologia , Streptococcus sanguis/genética , Proteínas de Bactérias/genética , Epitopos/genética , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Oligopeptídeos , Oxigênio/metabolismo , Peptídeos/genética , Piruvato Oxidase/genética , Streptococcus sanguis/crescimento & desenvolvimento , Transformação BacterianaRESUMO
The activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth-phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a sigma factor required to transcribe a number of stationary-phase-induced genes. PoxB activity as well as the beta-galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene. Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5' end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB. The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression. Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found that poxB null mutations had no effect on rpoS(katF) expression. Inactivation of two genes involved in acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Proteínas Ferro-Enxofre , Proteínas Quinases , Piruvato Oxidase/genética , Proteínas Repressoras , Fator sigma/metabolismo , Aerobiose/fisiologia , Sequência de Aminoácidos , Anaerobiose/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Sequência de Bases , Escherichia coli/enzimologia , Genes Bacterianos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Piruvato Oxidase/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
Two strains of Lactobacillus plantarum accumulated H2O2 when grown aerobically in a complex glucose based medium. The H2O2 accumulation did not occur immediately on exposure of the culture to O2 but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O2 utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H2O2 was not a product of NADH oxidase in vitro but was formed in substantial quantities from O2 during oxidation of pyruvate. The three enzymes were induced by O2 and H2O2; the induction of NADH oxidase responded to lower levels of O2 (but not of H2O2) than the pyruvate oxidase or the NADH peroxidase.