Paiment Anne, Hocking Jennifer, Whitfield Chris
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
J Bacteriol. 2002 Dec;184(23):6437-47. doi: 10.1128/JB.184.23.6437-6447.2002.
Wzc(CPS) is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the Wzc(CPS) C terminus is essential for HMW CPS assembly. Overexpression of Wzb(CPS) (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of Wzc(CPS) phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for Wzc(CPS) phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of Wzc(CPS) in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to Wzc(CPS) derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of Wzc(CPS). Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of Wzc(CA) from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30(CPS) from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that Wzc(CPS) forms complexes in vivo, independent of the phosphorylation state of the protein.
Wzc（CPS）是一种酪氨酸自激酶，对大肠杆菌中高分子量（HMW）1型荚膜多糖（CPS）的组装至关重要。Wzc的同源物参与多种革兰氏阳性和革兰氏阴性细菌中CPS和胞外多糖的形成。Wzc（CPS）C末端酪氨酸残基的磷酸化对于HMW CPS组装至关重要。在野生型背景中过表达Wzb（CPS）（磷酸酶）导致细胞相关K30 CPS产量下降3.7倍，证实了Wzc（CPS）磷酸化对荚膜组装的重要性。在本研究中，对富含酪氨酸的区域进行了剖析，试图鉴定对Wzc（CPS）磷酸化和/或荚膜表达至关重要的残基。定点诱变表明，该区域中没有单个酪氨酸残基足以在体内检测到Wzc（CPS）的磷酸化或HMW CPS表达。此外，没有单个酪氨酸残基对于磷酸化或荚膜组装是必不可少的，因为去除任何一个酪氨酸残基都没有可检测到的影响。改变酪氨酸残基的组合（从两个到五个）导致Wzc（CPS）衍生物仍然能够进行磷酸化，但不能支持HMW CPS的组装，表明Wzc本身的磷酸化不是其在荚膜组装中发挥功能能力的准确衡量标准。这些数据的一种解释是，该区域的整体磷酸化水平，而不是可磷酸化残基的精确组合，对Wzc（CPS）的活性很重要。酪氨酸569，一个已被证明可调节来自大肠杆菌K-12的Wzc（CA）体外磷酸化的残基，也被诱变。具有这种突变的衍生物仍然在荚膜组装中起作用。对该突变体的K30（CPS）进行定量分析，结果显示产生的聚合物量没有差异。最后，使用二硫代双（琥珀酰亚胺丙酸酯）交联来证实Wzc（CPS）在体内形成复合物，与蛋白质的磷酸化状态无关。
