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 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 （H202） able to antagonize cariogenic Strepto- coccus mutans （S. mutans）. Furthermore, the ecological consequence of H202 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 H202, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H202 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.展开更多
本文优化了表达绿色气球菌丙酮酸氧化酶的重组大肠杆菌的诱导条件。优化了诱导温度、诱导剂IPTG浓度、诱导时重组大肠杆菌的浓度、溶氧等条件后发现最佳的诱导条件为25℃、IPTG 0.5 mmol/L、OD_(600)0.5细胞浓度、250 m L摇瓶中50 m L...本文优化了表达绿色气球菌丙酮酸氧化酶的重组大肠杆菌的诱导条件。优化了诱导温度、诱导剂IPTG浓度、诱导时重组大肠杆菌的浓度、溶氧等条件后发现最佳的诱导条件为25℃、IPTG 0.5 mmol/L、OD_(600)0.5细胞浓度、250 m L摇瓶中50 m L发酵液。该条件下重组大肠杆菌诱导24 h后最终的丙酮酸氧化酶活力达到7 396.9 U/L,与优化前相比提高了4倍,是目前报道的最高水平。本研究结果为大肠杆菌大量表达有活性的丙酮酸氧化酶,以及丙酮酸氧化酶的生产应用奠定了基础。展开更多
基金supported by NIH grants 4R00DE 018400 to Jens Kreth
文摘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 （H202） able to antagonize cariogenic Strepto- coccus mutans （S. mutans）. Furthermore, the ecological consequence of H202 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 H202, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H202 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.
文摘本文优化了表达绿色气球菌丙酮酸氧化酶的重组大肠杆菌的诱导条件。优化了诱导温度、诱导剂IPTG浓度、诱导时重组大肠杆菌的浓度、溶氧等条件后发现最佳的诱导条件为25℃、IPTG 0.5 mmol/L、OD_(600)0.5细胞浓度、250 m L摇瓶中50 m L发酵液。该条件下重组大肠杆菌诱导24 h后最终的丙酮酸氧化酶活力达到7 396.9 U/L,与优化前相比提高了4倍,是目前报道的最高水平。本研究结果为大肠杆菌大量表达有活性的丙酮酸氧化酶,以及丙酮酸氧化酶的生产应用奠定了基础。
