Function of cell surface and degradation of xenobiotic polymers in Sphingomonas species

本研究从嗜聚乙二醇（PEG）和聚乙烯醇（PVA）鞘氨醇单胞菌（Sphingomonasmacrogoltabidus）103菌株中克隆了PEG脱氢酶（pegA）基因的上下游序列，并进行了序列测定。它包含10个推定基因：orf 1（A，B），peg B，C，D，A，E，R和orf 2（A，B），分别对应于转座酶（A，B），TonB依赖性受体，醛脱氢酶，通透酶，PEG脱氢酶（PEGDH），酰基辅酶A连接酶，AraC型转录调节因子（反向）和转座酶（A，B）。在另一种PEG降解菌S.macrogoltabidus菌株203和S. terrae中发现超过99%的基因结构（pegB、C、A、E和orf 2）同源性。该菌的Orf 1与巨金链霉菌103和203株的Orf 1同源性在50%左右（同源性均在90%以上，但方向相反）。Inse ...更多信息 在菌株203中发现pegD和pegA之间存在另一种转座酶。这导致pegA的组成型表达。斑点杂交和RT-PCR结果表明，5个基因在同一方向上转录为单个操纵子。pegD基因在大肠杆菌中表达，重组酶的特征在于尼古丁蛋白PEG-醛脱氢酶，其中NADP与该酶紧密结合。这是第一个发现尼古丁蛋白醛脱氢酶。另一方面，利用三维分子模拟技术对PEGDH的反应机理进行了研究，并对鞘氨醇单胞菌113 P3的氧化PVA水解酶（OPH）进行了纯化和表征。根据N端和内部氨基酸序列，克隆了OPH基因并进行了序列测定。重组酶在大肠杆菌中以包涵体形式表达。通过同源性搜索，OPH与假单胞菌VM 15 C和聚羟基丁酸酯解聚酶的OPH具有相似性，保持了氧阴离子空穴和催化三联体的共有序列。此外，编码PVA脱氢酶（PVADH）和细胞色素C的基因位于下游。OPH和PVADH基因几乎没有间隔地串联在一起，表明它们是由同一个启动子控制的共表达。利用PEG或PVA的鞘氨醇单胞菌在PEG或PVA培养基或营养肉汤中生长时，其细胞表面结构发生了变化，这似乎与它们的降解能力相对应。通过分析参与各自降解操纵子及其邻近的基因，我们可以显示聚合物是如何被识别并导致其代谢基因的表达的。TonB依赖性受体和通透酶似乎参与PEG降解中底物或代谢物的转运。这一点必须进一步澄清。少

In this study, biochemical and molecular studies on polyethylene glycol (PEG) and polyvinyl alcohol (PVA) degradation were practiced with PEG- or PVA-utilizing sphingomonads.The approximately 14-kb DNA fragment including upstream and downstream regions of a gene for PEG dehydrogenase (pegA) was cloned from Sphingomonas macrogoltabidus strain 103 and sequenced. It contained ten putative genes : orf1 (A,B), pegB,C,D,A,E and R and orf2 (A,B) corresponding to transposases (A,B), TonB-dependent receptor, aldehyde dehydrogenase, permease, PEG dehydrogenase (PEGDH), acyl-CoA ligase, AraC-type transcription regulator (in opposite orientation) and transposases (A,B), respectively. More than 99% homology of gene structures (pegB,C,A,E and orf 2) was found in the other PEG-degrader, S.macrogoltabidus strain 203 and S.terrae. Orf 1 of S.terrae had approximately 50% homology with those of S.macrogoltabidus strains 103 and 203 (homology of them are more than 90%, but the direction was reverse.) Inse … More rtion of another transposase between pegD and pegA was found in strain 203. which led to constitutive expression of pegA. Dot-blot hybridization and RT-PCR revealed that five genes in the same orientation transcribed as single operon. The gene, pegD was expressed in E.coli and the recombinant enzyme was characterized as nicotinoprotein PEG-aldehyde dehydrogenase including NADP tightly bound to the enzyme. This was the first finding on nicotinoprotein aldehyde dehydrogenase. On the other hand, reaction mechanism of PEGDH was characterized, based on 3D molecular modeling.Oxidized PVA-hydrolyzing enzyme (OPH) was purified from Sphingomonas sp.113P3 and characterized. Base on N-terminal and internal amino acid sequences, the gene encoding OPH was cloned and sequenced. The recombinant enzyme was expressed as inclusion body in E.coli. By homology search, OPH had similarity with OPH from Pseudomonas sp.VM15C and polyhydroxybutyrate depolymerases, keeping the consensus sequence for oxyanion hole and catalytic triads. In addition, the gene encoding PVA dehydrogenase (PVADH) and cytochrome C were located downstream. The genes for OPH and PVADH were located in tandem with almost no space, showing the coexpression controlled by the same promotor.PEG- or PVA-utilizing sphingomonads changed their cell surface structures when grown either on PEG or PVA-medium or on nutrient broth, which seemed to correspond to their degradation abilities. By analyzing genes involved in respective degradation operons and their vicinities we could show how polymers are recognized and lead to the expression of their metabolic genes. TonB-dependent receptor and permease seem to be involved in transport of substrates or metabolites in PEG degradation. This has to be clarified further. Less