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首页> 外文期刊>Applied and Environmental Microbiology >Functional Analysis of Burkholderia cepacia Genes bceD and bceF, Encoding a Phosphotyrosine Phosphatase and a Tyrosine Autokinase, Respectively: Role in Exopolysaccharide Biosynthesis and Biofilm Formation
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Functional Analysis of Burkholderia cepacia Genes bceD and bceF, Encoding a Phosphotyrosine Phosphatase and a Tyrosine Autokinase, Respectively: Role in Exopolysaccharide Biosynthesis and Biofilm Formation

机译：伯克霍尔德菌洋葱伯克霍尔德氏菌基因bceD和bceF的功能分析，分别编码磷酸酪氨酸磷酸酶和酪氨酸自身激酶：在胞外多糖生物合成和生物膜形成中的作用

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The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP), BceD exhibited a Vmax of 8.8 μmol of PNPP min?1 mg?1 and a Km of 3.7 mM PNPP at 30°C. The disruption of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.
机译：Burkholderia cepacia复合菌株生物合成胞外多糖（EPS）的pacapian需要基因簇16.2kb bce。 bceD和bceF这两个聚簇基因分别编码与磷酸酪氨酸磷酸酶和酪氨酸激酶同源的两种蛋白质。我们显示实验证据表明BceF在酪氨酸上被磷酸化，并且保守的赖氨酸残基存在于Walker A 563结合位置，此自磷酸化需要ATP结合基序。还证明了BceD能够使磷酸化的BceF脱磷酸。使用人工底物对硝基苯基磷酸酯（PNPP），BceD在30°C时的Vmax为8.8μmolPNPP min？1 mg？1，Km为3.7 mM PNPP。 bceF的破坏导致了培养基中cepacian积累的消失，但是bceD突变体仍记录了亲本菌株EPS产量的75％。由bceD突变体产生的胞外多糖产生的粘性溶液较少，并显示出与野生型cepacian相同的乙酰化程度，表明该突变体生物聚合物的分子量较低。由bceD和bceF突变菌株体外产生的生物膜的大小小于由亲本菌株形成的生物膜的大小，并且该表型通过互补测定法得以证实，表明BceD和BceF在生物膜的建立中发挥作用最大尺寸。

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