Overhage J, Kresse A U, Priefert H, Sommer H, Krammer G, Rabenhorst J, Steinbüchel A
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, D-48149 Münster, Germany.
Appl Environ Microbiol. 1999 Mar;65(3):951-60. doi: 10.1128/AEM.65.3.951-960.1999.
Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.
假单胞菌属菌株HR199能够利用丁香酚（4-烯丙基-2-甲氧基苯酚）、香草醛（4-羟基-3-甲氧基苯甲醛）或原儿茶酸作为唯一碳源进行生长。在用亚硝基胍诱变后，获得了该菌株的突变体，这些突变体在香草醛的分解代谢中受损，但仍保留利用丁香酚或原儿茶酸的能力。一个突变体（SK6169）用作携带黏粒pVK100的假单胞菌属菌株HR199基因组文库的受体，用一个5.8kbp的EcoRI片段（E58）实现了表型互补。从E58上鉴定出的两个相应开放阅读框（ORF）推导的氨基酸序列显示与编码原儿茶酸3,4-双加氧酶两个亚基的pcaG和pcaH具有高度同源性。另外三个ORF很可能编码一个4-羟基苯甲酸3-羟化酶（PobA）和两个推定的调节蛋白，它们分别与根癌土壤杆菌的PcaQ和铜绿假单胞菌的PobR具有同源性。由于突变体SK6169也被仅含有pcaH的E58亚片段互补，这个突变体很可能缺少原儿茶酸3,4-双加氧酶的功能性β亚基。由于这个突变体仍能够在原儿茶酸上生长且缺乏原儿茶酸4,5-双加氧酶和原儿茶酸2,3-双加氧酶，降解必定由不同的酶催化。另外两个在香草醛分解代谢中也受损的突变体（SK6184和SK6190）未被片段E58互补。由于这些突变体在丁香酚培养过程中积累3-羧基粘康酸内酯，它们很可能在邻位裂解后的分解代谢途径的某一步表现出缺陷。此外，在这些突变体中，3-羧基粘康酸的环化似乎通过顺式绝对立体化学过程发生，这通常仅在假单胞菌属中顺式,顺式-粘康酸内酯化中观察到。总之，在假单胞菌属菌株HR199中香草醛通过邻位裂解途径降解，而在突变体中原儿茶酸也可通过不同途径代谢。
