Role of operon aaoSo-mutT in antioxidant defense in Streptococcus oligofermentans.
Previously, we have found that an insertional inactivation of aao(So), a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aao(So) and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aao(So) and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aao(So) insertional mutant, indicating that gene polarity derived from the inactivation of aao(So) attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H(2)O(2) (min · mg protein)(-1)]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aao(So) mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant's growth by 11%, indicating that the aao(So) mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aao(So). Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aao(So) mutant. This demonstrated that inactivation of aao(So) caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aao(So)-mutT operon in antioxidant defense in S. oligofermentans.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Peng
P
Zhou
Lei
L
Liu
Huichun
H
Tong
Xiuzhu
X
Dong
D000096:Acetone / Q000031:analogs & derivatives / Q000378:metabolism; D000975:Antioxidants / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D020868:Gene Silencing; D050830:L-Amino Acid Oxidase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon / Q000235:genetics*; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013291:Streptococcus / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
D000975:Antioxidants; D001426:Bacterial Proteins; D009711:Nucleotides; D017382:Reactive Oxygen Species; D000096:Acetone; C095500:SodA protein, Bacteria; D013482:Superoxide Dismutase; D050830:L-Amino Acid Oxidase; D011755:Pyrophosphatases; C006495:aminoacetone