Chee-Sanford J C, Frost J W, Fries M R, Zhou J, Tiedje J M
Department of Microbiology, Michigan State University, East Lansing 48824, USA.
Appl Environ Microbiol. 1996 Mar;62(3):964-73. doi: 10.1128/aem.62.3.964-973.1996.
A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.
一种甲苯降解反硝化菌，解甲苯偶氮弧菌Tol-4，是从三个受石油污染的含水层沉积物中分离出的八株类似菌株之一。当该菌株在甲苯上厌氧生长时，发现甲苯中68%的碳以二氧化碳形式存在，30%以生物量形式存在。菌株Tol-4的倍增时间为4.3小时，最大反应速度（Vmax）为50微摩尔·分钟-1·克蛋白质-1，细胞得率为49.6克·摩尔甲苯-1。苯甲酸似乎是一种中间产物，因为氟苯甲酸从氟甲苯中积累，并且在过量苯甲酸存在的情况下，[14C]苯甲酸由[14C]甲苯产生。两种代谢产物，E-苯基衣康酸（1%至2%）和苄基琥珀酸（<1%），从厌氧甲苯代谢中积累。当细胞在氢化肉桂酸和反式肉桂酸上生长时也会产生这些相同的产物，但在苄醇、苯甲醛、苯甲酸、对甲酚或它们的羟基化类似物上生长时不会产生。这些证据支持了一种厌氧甲苯降解途径，该途径涉及菌株Tol-4中最初的乙酰辅酶A（乙酰-CoA）攻击，正如埃文斯及其同事（P. J. 埃文斯、W. 凌、B. 戈德施密特、E. R. 里特和L. Y. 杨，《应用与环境微生物学》58:496 - 501, 1992）针对另一种甲苯降解反硝化菌T1菌株所提出的那样。我们的发现支持对所提出途径的一种修改，即肉桂酰-CoA在氢化肉桂酰-CoA氧化之后产生，类似于推测的苄基琥珀酸氧化形成E-苯基衣康酸。在存在甲苯和[14C]乙酸盐的情况下生长的Tol-4培养物中检测到了肉桂酸。我们进一步提出，肉桂酰-CoA添加第二个乙酰-CoA是苄基琥珀酸和E-苯基衣康酸的来源。这一途径得到以下发现的支持：添加到甲苯生长培养物中的单氟乙酸盐导致苄基琥珀酸和E-苯基衣康酸的产量显著增加，以及在细胞与甲苯、[14C]乙酸盐和肉桂酸一起孵育后检测到[14C]苄基琥珀酸。未发现通过甲基氧化进行厌氧甲苯代谢的证据，因为在与苄醇和苯甲醛一起孵育后未检测到苄基琥珀酸和E-苯基衣康酸，甚至在14C捕获实验中也未检测到苄醇和苯甲醛。
